Saccharomyces cerevisiae reduces vulvovaginal candidiasis severity through modulation of fungal pathogenicity and inflammatory responses

Saccharomyces cerevisiae reduces vulvovaginal candidiasis severity through modulation of fungal pathogenicity and inflammatory responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Saccharomyces cerevisiae reduces vulvovaginal candidiasis severity through modulation of fungal pathogenicity and inflammatory responses Mart Sillen, Dania El Abyad, Nina Vreys, Silke Baldewijns, Ilse Palmans, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6795973/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Vulvovaginal candidiasis (VVC) affects millions globally and is characterized by multifactorial immunopathology, with Candida albicans virulence driving disease progression through epithelial tissue damage and neutrophil hyperactivation and dysfunction contributing to disease severity. Here, we explored the multifaceted nature of S. cerevisiae as a live-biotherapeutic to attenuate C. albicans virulence and modulate host immune responses during VVC. We identified an S. cerevisiae isolate that targets multiple aspects of C. albicans virulence, including fungal proliferation, adhesion, and hyphal morphogenesis, collectively impairing biofilm formation and disrupting pathogenic potential. These effects were linked to transcriptional reprogramming in C. albicans , marked by metabolic stress and downregulation of virulence- and biofilm-related genes. Additionally, S. cerevisiae reduced inflammatory responses and neutrophil hyperactivation, while preserving neutrophil antimicrobial functionality. Collectively, this translated to an improved control of infection and dampened VVC-associated hyperinflammation in a murine VVC model. These findings position S. cerevisiae as a promising live biotherapeutic to mitigate C. albicans infection and alleviate hyperinflammation in VVC. Further clinical validation in human cohorts is essential to confirm its therapeutic efficacy. One Sentence Summary: S. cerevisiae attenuates C. albicans virulence and inflammation, offering therapeutic potential for vulvovaginal candidiasis. Health sciences/Diseases/Infectious diseases/Fungal infection Biological sciences/Microbiology/Fungi/Fungal host response Biological sciences/Microbiology/Cellular microbiology Candida albicans biofilm formation neutrophil activation bulk RNA-seq epithelial infection VVC mice model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Vulvovaginal candidiasis (VVC), an infection of the vaginal mucosa caused by yeast of the genus Candida , is the second most prevalent form of vaginitis, posing a significant health concern 1 , 2 . VVC affects up to 75% of women at least once, regardless of their overall health status 3 . Moreover, approximately 7–9% of women endure recurrent vulvovaginal candidiasis (RVVC), defined by three or more infections annually 4 , 5 , contributing to an estimated global burden of 138 million cases per year 6 . (R)VVC manifests distressing symptoms, such as white clumpy discharge, burning, redness, and itching in the vulva and vagina, and dyspareunia, significantly impacting mental well-being 3 , 7 . Candida albicans , the most prevalent causative species 8 , 9 , induces VVC through hyperinflammation, driven by Candida virulence and maintained by the innate immune system, resulting in compromised neutrophil-mediated fungal clearance 8 , 10 . VVC manifests when the fungal burden exceeds an epithelial threshold, triggering epithelial pro-inflammatory signaling cascades that result in the release of inflammatory mediators 8 . This process catalyzes downstream inflammatory responses that facilitate the recruitment of immune cells, primarily neutrophils 11 . However, the hyperinflammation and specific characteristics of the vaginal niche impair neutrophils' ability to effectively deploy their antifungal mechanisms, leading to tissue damage and perpetuating a cycle of inflammation and exacerbation of symptoms 11 . Being a multifaceted disease, VVC presents multiple therapeutic opportunities, including antifungal drugs and vaccines still under research 3 , 12 . While antifungals are central to RVVC management, prolonged use often fosters resistance 4 , 5 . Coupled with the complex nature of VVC, this leaves many patients insufficiently treated. Moreover, antifungals only indirectly impact inflammatory responses and fail to alleviate symptoms when non-self-limiting inflammation drives disease progression 13 . Therefore, alternative treatment strategies that tackle VVC’s multifactorial nature are urgently needed. Given the essential role of vaginal microbial communities in maintaining vaginal health, live microbial-biotherapeutics present a promising avenue 14 , 15 . Therefore, alternative treatment strategies that tackle VVC’s multifactorial nature are urgently needed. Given the essential role of vaginal microbial communities in maintaining vaginal health, live microbial-biotherapeutics (LBPs) present a promising avenue 14 , 15 . These are defined as live microorganisms intended for the prevention or treatment of disease 16 , and several Lactobacillaceae -based LBPs have shown efficacy in experimental and clinical VVC 17 , 18 . However, as infections are highly patient-specific, treatment efficacy is not universal 19 . Although yeast are generally not dominant members of the vaginal microbiome, clinical research highlights reduced levels of Saccharomyces spp. in women with RVVC compared to healthy individuals, making these yeasts intriguing candidates for further study 20 . S. cerevisiae especially shows promise for use in patients undergoing antibiotic therapy, a common trigger for VVC development 2 . Recognized for its benefits in gastrointestinal disorders 21 , 22 , one S. cerevisiae strain has also shown efficacy in the context of VVC, demonstrating the ability to coaggregate with C. albicans , inhibit its adherence to epithelial cells, suppress key virulence factors, and protect epithelial cells from Candida -induced damage 23 , 24 . Its effects likely arise from mechanisms such as pathogen binding, production of antimicrobial peptides, trophic effects, and immune-modulatory properties 22 , 25 . In this study, we focused on the potential of S. cerevisiae in counteracting C. albicans -induced VVC in lab-based and murine models. We conducted a first-in-disease comprehensive screening of a broad library of S. cerevisiae isolates to assess their potential as therapeutic microbes against VVC. We explored the mechanisms through which S. cerevisiae antagonizes C. albicans pathogenicity and modulates immune responses, focusing on insights gained from multispecies transcriptomics combined with in vitro infection models. To validate these findings in a physiologically relevant context, we treated mice with S. cerevisiae in the context of a well-established VVC model. Through examining the tripartite interactions between C. albicans , vaginal epithelial cells, and neutrophils—both in in vitro and in murine models—we uncovered novel mechanistic insights into how S. cerevisiae acts as a live biotherapeutic for VVC. C. albicans virulence inhibition by physical constraint, metabolic interference and transcriptional reprogramming resulted in a dampened pro-inflammatory milieu and enhanced neutrophil-mediated fungal clearance, two critical determinants in the pathogenesis of symptomatic VVC. These findings establish a robust preclinical foundation for the translational development of S. cerevisiae -based interventions in human VVC. Results S. cerevisiae strain-specific inhibition of C. albicans . Given large strain-to-strain variability within microbial species 19 , 26 , a screening was performed to identify S. cerevisiae strains with inhibitory properties against C. albicans , a key driver of VVC pathology 10 . We screened 70 diverse S. cerevisiae strains, including food, lab strains, probiotics, beverages, and human sources ( Table S1 ). Using assays for C. albicans CA3153 growth inhibition in vagina-simulative medium (VSM) and RPMI, adhesion to A-431 vaginal epithelial cells (VECs), and filamentation inhibition, several effective S. cerevisiae strains were identified ( Fig. S1 ). Notably, inhibitory capacity was not linked to the strain's origin. From the 70 strains initially screened, the most promising candidates were shortlisted based on top performance in inhibiting C. albicans virulence, with those showing consistent efficacy across multiple assays prioritized. Based on these criteria, strains 4609, 6883, 6886, 6888, 6894, 3458, 3471, 3076, and Y.00240 were selected ( Table S1 ). Two additional control strains were also included: the lab strain S288c and the probiotic standard CNCM I-3856, currently marketed for gastrointestinal ( ibSium® ) and vaginal ( Quatreflora® ) health 24 , 27 . S. cerevisiae reduces C. albicans pathogenicity on multiple levels. To investigate the inhibitory potential of S. cerevisiae against C. albicans virulence in depth, we built on our extensive screening and focused on early infection processes across three C. albicans isolates SC5314, CA3153, and I49. Growth of C. albicans isolates in both VSM (Fig. 1 A, 1 B) and RPMI ( Fig. S2A ) was broadly inhibited by the selected S. cerevisiae strains, reducing C. albicans growth by up to 25–50% within 6 hours and reaching up to 75% inhibition by 24 hours. Notably, specific strains - C(6886), F(3458), G(3471), and IC(CNCM I-3856) - achieved higher levels of inhibition at earlier time points. Moreover, the growth inhibition was robust across all tested C. albicans isolates. The adhesion of C. albicans to VECs is crucial for the initiation of infection, invading the epithelial cells and causing damage 28 . Simultaneous introduction of S. cerevisiae reduced C. albicans adhesion to VECs by ~ 30%, while some strains displaced up to 50% of pre-adhered C. albicans cells (Fig. 1 C), disrupting established C. albicans –VEC interactions. Despite variability between S. cerevisiae strains and individual repetitions, certain strains consistently inhibited adherence of all three C. albicans isolates. The morphological transition to filamentous growth strengthens adhesion of C. albicans and facilitates invasion and damage of epithelial cells 28 . This further prompts C. albicans recognition and initiation of epithelial inflammatory responses 8 , 29 . Specific S. cerevisiae strains (A(4609), C(6886), F(3458), G(3471), and H(3076)) were able to inhibit (30–50%) or delay hyphal growth in VSM after 3 hours (Fig. 1 D), though the effects were less pronounced in RPMI which by itself is a stronger stimulus for filamentation ( Fig. S2B ). While all S. cerevisiae strains reduced C. albicans growth in VSM (Fig. 1 A, B), not all inhibited the morphological transition, and some, B(6883), I(Y.00240), and LC(S288c), even promoted hyphal growth. S. cerevisiae mitigates C. albicans -induced epithelial damage and inflammation. The inhibitory effect of S. cerevisiae on C. albicans growth, filamentation and adhesion to VECs could imply decreased epithelial tissue damage and inflammatory responses 29 . S. cerevisiae co-cultured reduced epithelial damage inflicted by C. albicans , quantified by the release of the host cytoplasmic enzyme LDH (Fig. 2 A, B). While certain S. cerevisiae strains - D(6888), E(6894), F(3458), G(3471) - markedly reduced cell damage, some - I(Y.00240), LC(S288c), and IC(CNCM I-3856) - had minimal effect or even exacerbated toxicity. Through the release of alarmins such as IL-1α, epithelial tissue damage can catalyze a pro-inflammatory state, activating neutrophils 8 . C. albicans infection alone triggered minimal IL-1α release (Fig. 2 A, C), which was not significantly altered by protective S. cerevisiae strains, suggesting that additional factors may regulate its production. MAPK pathways, activated by excessive fungal burden and hyphae, exacerbate VVC pathology by recruiting and activating neutrophils 11 , 29 , 30 . S. cerevisiae strains that dampened fungal growth and filamentation also reduced IL-8 and GM-CSF responses in VECs (Fig. 2 D, E), cytokines that enhance neutrophil activation, survival, and effector function (Fig. 1 C). Importantly, the probiotic S. cerevisiae strain IC (CNCM I-3856) enhanced pro-inflammatory cytokine responses, amplifying signaling beyond the response to C. albicans . Unlike other S. cerevisiae strains, it also independently triggered pro-inflammatory responses in VECs in the absence of C. albicans ( Fig. S3 ). Cluster analysis reveals superior S. cerevisiae strains for VVC treatment. Our data demonstrate that S. cerevisiae strains can variably attenuate C. albicans virulence and alleviate the epithelial immune responses. We aimed to identify a strain that consistently exhibited superior performance across all assays. Multivariate cluster analysis using the collective data from previous assays revealed two distinct clusters (k-means, k = 2), explaining over 70% of the variance observed in the dataset (Fig. 2 F, G). This significant level of explained variance indicates a strong association between the identified clusters and the observed performance outcomes. The first cluster, comprised of the S. cerevisiae strains that demonstrated superior efficacy in reducing C. albicans virulence and modulating immune responses, included A(4609), B(6883), C(6886), E(6894), F(3458), G(3471), and H(3076), suggesting a robust live biotherapeutic potential. The second cluster comprised of strains that exhibited overall low performance: D(6888), I(Y.00240), LC(S288c), and IC(CNCM I-3856). Based on the consistently high performance across all readouts, S. cerevisiae strain (F) 3458 (Sc3458) was selected for further investigation. S. cerevisiae drives transcriptional reprogramming in C. albicans and VECs. Multispecies transcriptomics was performed to identify underlying mechanisms by which Sc3458 attenuates C. albicans virulence and modulates epithelial inflammatory responses. Principal component analysis (PCA) revealed distinct C. albicans gene expression at 24-hours post-infection (hpi) with slight overlap at 6 hpi, depending on the presence of Sc3458 (Fig. 3 A). Similarly, VECs showed distinct clusters in the presence of Sc3458 at 24 hpi (Fig. 3 B). Despite the proximity of PCA clusters, particularly for C. albicans at 24 hpi, significantly differentially expressed genes (DEGs) were identified (Fig. 3 C, upper panel ). PCA highlights major variability in gene expression between conditions, but overlapping clusters can still harbor substantial transcriptional differences. At 6 hpi, C. albicans showed 9 DEGs ( Fig. S4 ), but this increased to 211 DEGs at 24 hpi in response to Sc3458. For VECs, the impact of S. cerevisiae was more pronounced at 24 hpi, with 1848 DEGs observed (Fig. 3 C, lower panel ). Together, the DEG counts and PCA results underscore that Sc3458 drives significant reprogramming of both C. albicans and VEC gene expression, particularly at 24 hpi. More specifically for C. albicans , a total of 131 genes were downregulated, while 80 genes were upregulated when S. cerevisiae was present during infection (Fig. 3 D). Gene Ontology (GO) enrichment analysis revealed upregulation of genes related to “amino acid transport and metabolism” ( AGP2, ALP1, SER2 ) and “sulfur compound biosynthesis” ( SAM2, MET16, MET15, CYS3 ), reflecting metabolic shifts to counter nutrient competition and oxidative stress (Fig. 3 F). Increased expression of ion transport genes ( FRE10, CFL2, CFL5 ) suggests efforts to maintain ionic and redox balance under oxidative stress. Upregulated biosynthetic ( ACS2, ACC1 ) and ribosomal genes ( GAR1, RPL39 ) indicate heightened protein synthesis demands to manage stress and support growth. These stress-induced adaptations highlight C. albicans' attempt to reprogram its metabolism and homeostatic mechanisms to survive the antagonistic effects imposed by Sc3458 during infection. The analysis of downregulated genes revealed links to processes essential for C. albicans biofilm development and cell-cell interaction (Fig. 3 E). GO term enrichment identified significant downregulation in genes involved in “single-species biofilm development” (both submerged and on inanimate substrates), “cell aggregation,” and “intraspecies interactions.” Key metabolic pathways for nutrient acquisition within the biofilm microniche were impacted, with genes involved in monosaccharide, hexose, and carbohydrate transport ( HGT13, GAL1, MAL31 ) downregulated, suggesting compromised biofilm integrity and cellular function. Genes related to adhesion ( TRY5, SUC1, ZCF28, SAP2/4/8, DSE1 ), morphogenesis ( HGC1, YAK1, CSA2, MAL2 ), and biofilm maturation ( SAP8, PGA6 ) were also suppressed, proposing significant disruption of biofilm structure and functionality. Direct interaction and metabolic activity of S. cerevisiae are essential for anti-biofilm effects on C. albicans. C. albicans biofilm formation on epithelial cells during VVC exceeds the epithelial threshold, triggering inflammatory responses and hindering neutrophil-mediated fungal clearance 30 , 31 . While metabolic changes in C. albicans induced by probiotics are known to reduce pathogenicity 32 , their specific effects on biofilm formation remain unclear. As biofilms play a key role in VVC pathogenesis, we investigated how Sc3458 impacts C. albicans biofilm formation. Corresponding with reduced C. albicans growth and filamentation on VECs (Fig. 1 ), a broad downregulation of C. albicans biofilm-related genes in response to the presence of Sc3458 is observed (Fig. 4 A). Accordingly, Z-stack microscopy proved that Sc3458 significantly reduced C. albicans biofilm biomass on VECs during both early (24 hpi) and mature biofilm (48 hpi) stages (Fig. 4 B, C). Additionally, biofilm thickness was diminished by Sc3458 (Fig. 4 B, D). These inhibitory effects were comparable to those observed with the industrial control CNCM I-3856. We hypothesized that S. cerevisiae might redistribute C. albicans cells between the biofilm associated with VECs and cells in the supernatant, given the known aggregation tendencies of both species 33 . Interestingly, redistribution of cells from the biofilm to the supernatant was observed upon Sc3458 treatment (Fig. 4 E). A similar redistribution pattern was observed for IC (Fig. 4 E), suggesting a conserved interference mechanism. While Sc3458 alone primarily remained in the supernatant, indicating weak adhesion to VECs (Fig. 4 F), the presence of C. albicans resulted in its incorporation into the biofilm. Strikingly, Sc3458 treatment displaced C. albicans into the supernatant while embedding itself within the C. albicans biofilm, resulting in fewer C. albicans cells being directly associated with the VECs and capable of inflicting damage and eliciting inflammatory responses. Given the gene expression patterns in C. albicans indicating cell aggregation and metabolic stress, we investigated whether the inhibitory effects of Sc3458 depend on direct contact and metabolically active cells. Using transwells to separate Sc3458 from C. albicans , allowing nutrient and molecule exchange, completely abolished the inhibition of biofilm formation (Fig. 4 G). Similarly, heat-inactivated Sc3458 cells failed to inhibit biofilm formation (Fig. 4 G). These findings indicate that biofilm inhibition by Sc3458 depends on both direct contact and cellular metabolism. The IC strain showed similar contact-dependent behavior (Fig. 4 G), reinforcing the robustness of this mechanism (Fig. 4 H). S. cerevisiae suppresses pro-inflammatory responses and neutrophil activation. To assess how Sc3458 treatment modulates inflammatory responses, we conducted a detailed analysis of the epithelial transcriptional changes, revealing significant reprogramming at 24 hpi (Fig. 3 B, C). A total of 1581 genes were downregulated and 267 upregulated (Fig. 5 A). GO enrichment analysis identified enhanced biological processes, including mitochondrial function, DNA repair, and RNA transport and processing, in response to Sc3458 treatment (Fig. 5 C). Although direct and well-established links to VVC pathology remain elusive, the GO-terms suggest a general activation of integral cellular maintenance and adaptive response pathways. The GO enrichment analysis highlighted many downregulated processes ( Fig. S5 ). We focused particularly on those relevant in the context of VVC, such as responses to stimuli, cell death, and inflammatory responses (Fig. 5 B). Key affected pathways included “cell surface receptor signaling”, “environmental and abiotic stimulus responses”, and “regulation of apoptotic and inflammatory signaling”. Notably, critical immune pathways —Nuclear Factor kappa B (NF-κB), Mitogen-Activated Protein Kinase (MAPK), and Phosphoinositide 3-Kinase/Protein Kinase B (PI3K/Akt) cascades— were downregulated, alongside Wnt signaling, phagocytosis, and autophagy regulation. Given the importance of MAPK, NF-κB, and PI3K/Akt pathways in epithelial responses to C. albicans 30 , 34 , we further analyzed specific DEGs within these pathways. Key genes in the MAPK pathway ( MKK7 and MLK3 ) were downregulated, critical for activating JNK and p38 ( Fig. S6 ) 35 . MK2 and ICAM1 , involved in inflammatory signaling, were also downregulated. MK2 regulates cytokine production via the p38 MAPK pathway, while ICAM1 facilitates immune cell adhesion and recruitment 36 . ERK1 , part of a separate MAPK branch, was also downregulated, a pathway associated with processes such as cell proliferation and stress responses to C. albicans 37 . In the NF-κB pathway, p65 , RelB , and NEMO were all downregulated, components essential for cytokine transcription and anti-apoptotic signaling 38 . Similarly, AKT1 , AKT2 , and IRS1 , part of the PI3K/Akt pathway, were downregulated, affecting survival signaling 39 . Reduced expression of TLR2 and FGFR1 / 4 indicated diminished immune activation and growth factor signaling. Downregulation of eNOS , FOXO1 , and FOXO4 further supports a shift in apoptosis regulation 40 . Transcriptional profiling revealed upregulation of IL1B in C. albicans -infected VECs exposed to S. cerevisiae ( Fig. S6 ). Excessive IL-1β production hyperactivates neutrophils, causing tissue damage without effectively clearing the pathogen, a hallmark of severe VVC. Unlike macrophages, VECs produce limited inflammasome-dependent IL-1β, and no IL-1β responses have been detected upon C. albicans infection of VECs 41 . By assessing IL-1β release by human monocyte-derived macrophages (hMDMs) 8 , we found that Sc3458 rather reduced IL-1β responses (Fig. 5 D). In contrast, the IC strain increased IL-1β secretion (Fig. 5 D), indicating that it reinforces inflammation across both epithelial (Fig. 2 ) and macrophage compartments. These data highlight a key functional distinction: while IC broadly amplifies inflammation, Sc3458 appears to reduce inflammation across epithelial cells and macrophages. Next, we validated how the downregulation of pro-inflammatory MAPK and NF-κB pathways corresponded to reduced downstream neutrophil activation by the epithelial cells. Supernatants from epithelial infections with Sc3458 led to slightly lower IL-8 (Fig. 5 E,F ) secretion and reduced ROS release by neutrophils (Fig. 5 G). Both effects were also seen with the IC strain, albeit with strain-specific variability. Although not all changes were statistically significant, an overall trend of reduced neutrophil activation was evident. In line with this, neutrophils exposed to supernatants from C. albicans -infected VECs treated with Sc3458 exhibited altered surface marker expression (Fig. 5 H). CD11b, an integrin involved in fungal recognition and recruitment, was downregulated, alongside CD66b, a marker of secondary granule mobilization. CD35 also decreased, suggesting reduced degranulation. In contrast, CD16 expression was elevated, consistent with enhanced neutrophil viability or antimicrobial readiness. Notably, CD62L, typically shed during activation, was preserved, indicating a less activated phenotype. Moderate reduction in CXCR2, which is internalized upon chemokine engagement, pointed to recent activation and potential migratory readiness. These expression patterns were broadly comparable to those induced by the IC strain, suggesting that, despite differences in upstream modulation of inflammation, both strains elicit a similarly tempered neutrophil phenotype. Nevertheless, responses varied depending on the C. albicans isolate used, with CA3153 and I49 generally inducing weaker pro-inflammatory signaling. Importantly, Sc3458 and IC did not affect neutrophil chemotaxis across the endothelial–epithelial barrier in response to C. albicans -infected VECs (Fig. 5 I). Collectively, these findings indicate that Sc3458 modulates neutrophil activation by dampening upstream epithelial signaling, while preserving neutrophil recruitment to the site of infection. S. cerevisiae promotes neutrophil-mediated C. albicans clearance. Given that Sc3458 reduced neutrophil activation downstream of C. albicans -infected VECs, we next assessed its direct effects on neutrophil-mediated fungal clearance, known to be impaired during VVC 8 . In addition to inhibiting C. albicans biofilm formation (Fig. 4 ), Sc3458 treatment enhanced neutrophil-mediated clearance of C. albicans biofilm-associated cells (Fig. 6 A). This led to an additive effect, with C. albicans biofilm biomass reduced by 30–50% beyond the inhibition observed with Sc3458 treatment alone. To further evaluate this interaction, we assessed neutrophil-mediated killing at early time points. Sc3458 significantly enhanced fungal killing at both 1 and 3 hpi, reducing fungal viability up to ~ 40% compared to neutrophils alone (Fig. 6 B). A similar enhancement of neutrophil-mediated clearance was observed with strain IC, suggesting that this trait can be shared across S. cerevisiae strains. The enhanced killing was consistent for C. albicans isolates SC5314 and CA3153. However, for isolate I49, which previously showed reduced virulence and delayed immune activation, the neutrophil-mediated killing effect was absent at 1 hpi. Using live-cell analysis, we simultaneously assessed fungal overgrowth, neutrophil death and NETosis (Fig. 6 C). Quantification of mScarlet-tagged C. albicans revealed significant reductions in C. albicans burden upon Sc3458 and IC treatment overtime, starting 2 hpi (Fig. 6 D). The visualization of treatment after 6 hours helped us to understand the interactions between neutrophils and C. albicans , in the presence of S. cerevisiae (Fig. 6 C). Images show enhanced neutrophil clustering and reduced C. albicans overgrowth upon Sc3458 treatment compared to neutrophils cultured with C. albicans alone, where fungal persistence was more evident. Interestingly, improved C. albicans killing and reduced overgrowth were not accompanied by significant changes in overall neutrophil cell death (Fig. 7 S). Similarly, NETosis events were not enhanced by the presence of Sc3458 or strain IC (Fig. 6 E), suggesting that enhanced fungal clearance occurs through mechanisms independent of NETosis. S. cerevisiae 3458 reduces C. albicans burden and attenuates local inflammation in a murine model. To determine whether the pathogenicity attenuating and immunomodulatory effects observed in vitro translated to an improved disease outcome, we treated mice with Sc3458 in the context of a well-established murine VVC model. Mice in a pseudoestrous state were intravaginally administered Sc3458, the industrial control strain (IC), or PBS (10 µL of 10⁸ cells/mL per mouse) one day after infection with C. albicans (10 µL of 10⁷ cells/mL per mouse) (Fig. 7 A). Longitudinal bioluminescence imaging (BLI) of the bioluminescent C. albicans reporter revealed a significant reduction in fungal burden following treatment with Sc3458. From day 6 onward, mice treated with Sc3458 exhibited a sustained decrease in C. albicans vaginal burden, while PBS-treated controls increased in fungal burden (Fig. 7 B, C; Fig. S8 ). The IC strain showed a slight early reduction in fungal burden compared to C. albicans alone; however, this trend was not statistically significant and was not sustained beyond day 6, highlighting a lack of durable antifungal activity compared to Sc3458 (Fig. 7 B, C). Along with the high fungal burden in PBS-treated controls 10-days post infection we observed an IL-1-(IL-1α and IL-1β) dominated inflammatory response in vaginal lavages that has been extensively associated with the dysregulated inflammatory state driving VVC severity (Fig. 7 D). Sc3458 treated mice, however, did not exhibit this characteristic pro-inflammatory response. Given the 10-day infection, adaptive immune responses are also activated. Even though controversial regarding their contribution to VVC 8 , treatment with Sc3458 dampened both Th17- and Th22-associated cytokines IL-17A and IL-22 (Fig. 7 D). Furthermore, type 2 immunity- and allergy-related cytokines, including IL-4, IL-9, IL-31, and the eosinophil-recruiting chemokine CCL11 (eotaxin-1), were significantly reduced in the Sc3458-treated group (Fig. 7 D, Fig. S8 ), suggesting a potential decrease in mucosal irritation and symptom severity. Interestingly, while most inflammatory mediators were diminished in the vaginal niche following Sc3458 treatment, neutrophil-chemokines such as CXCL2 remained elevated and CXCL1, and CXCL5 were even significantly higher (Fig. 7 D, Fig. S9 ), indicating a preserved or possibly enhanced chemotactic signal for neutrophil recruitment. Whilst the industrial control also decreased levels of several inflammatory mediators, similar to our observation in vitro , it increased IL-1β release and showed an even stronger type 2 phenotype reflected in IL-4 and CCL11 levels. These observations reaffirm the unique immunomodulatory potential of Sc3458 compared to the IC strain (Fig. 7 D, Fig. S9 ). Flow cytometry of vaginal lavage samples revealed minimal differences in total neutrophil and macrophage counts across infected groups (Fig. 7 E), indicating that immune cell recruitment was not substantially altered by treatment. However, only Sc3458-treated mice exhibited reduced fungal burden, suggesting enhanced neutrophil functionality despite similar cell numbers. Eosinophil counts remained stable, yet Sc3458 downregulated CD11b and CD11c expression on eosinophils (Fig. 7 F). While these integrins are often used as activation markers, they also play direct roles in pathogen recognition and cytokine signaling in eosinophils 42 . The observed downregulation is consistent with reduced type 2 inflammatory cytokines and may reflect a shift toward a less activated, less tissue-damaging phenotype. Discussion This study establishes the therapeutic potential of a specific S. cerevisiae strain in the context of C. albicans -driven VVC. Through systematic screening, we identified S. cerevisiae 3458, originally isolated from the vaginal mycobiota, as highly capable of inhibiting C. albicans growth, adhesion, and hyphal morphogenesis. This strain further set itself apart in the ability to reduce epithelial damage and inflammatory responses. Transcriptomic profiling and in vitro infection models revealed that Sc3458 attenuates C. albicans biofilm formation and suppresses pro-inflammatory epithelial signaling (MAPK, NF-κB). Additionally, it enhanced neutrophil-mediated fungal clearance in vitro . In a murine model of VVC, treatment with Sc3458 significantly reduced fungal burden while steering from IL-1-driven hyperinflammation and type-2 allergic responses. Collectively, this underscores Sc3458’s translational relevance as a live biotherapeutic candidate. Effective and affordable treatments for VVC are urgently needed due to its high prevalence and the increasing incidence of treatment failures driven by rising antifungal resistance 6 , 43 , 44 . Nevertheless, a universal cure is challenging due to heterogeneous predisposing factors, including genetic predisposition affecting immune responses, C. albicans virulence, and personal habits 45 . Despite this complexity, the prevailing view is that while hyperactivation of the epithelial cells and neutrophils play a major role in the onset of VVC, C. albicans virulence is equally crucial in driving disease progression 10 . To address these multifactorial drivers, we explored S. cerevisiae as a live biotherapeutic targeting multiple VVC-related factors 23 , 46 . We maintained consistent S. cerevisiae -to- C. albicans ratios, creating conditions that reflect a balanced interaction rather than overwhelming dominance, allowing us to evaluate its therapeutic efficacy under more equal conditions. S. cerevisiae has proven itself as a promising probiotic across diverse niches, combating pathogens and modulating immune responses 23 , 25 , 46 , 47 . However, our screening revealed that many previously characterized probiotic isolates were ineffective as live biotherapeutics against C. albicans in the VVC context. No clear correlation between niche specificity and antifungal activity emerged, likely due to strain-specific differences shaped by habitat-related idiosyncrasies, such as the microbial environment, host factors, and nutrient availability 48 , 49 . While the two most effective isolates, 3458 and 3471, originated from vaginal environments, other vaginal isolates showed limited efficacy. This underscores that habitat origin alone does not predict performance as a live biotherapeutic, reflecting the complexity of strain-specific adaptations. Sc3458 potently attenuated multiple C. albicans virulence traits, including proliferation, adhesion to epithelial surfaces, and the yeast-to-hypha transition. Given the multifactorial nature of effects against C. albicans and inflammation, it is unlikely that a single mechanism underlies this inhibition. Although early time points like 6 hpi could capture direct responses, only a small number of genes were differentially expressed, and these are not established virulence factors. Genes such as CSA2 , ADH2 , and FMN1 are primarily involved in metabolic or housekeeping functions 50 , 51 . In contrast, the broader transcriptional changes at 24 hpi, while potentially shaped by secondary effects, offer more insight into the cellular response to S. cerevisiae treatment and highlight relevant functional pathways. Metabolic interference, previously described for lactobacilli, is a plausible contributor 32 , 52 . Transcriptomic profiling revealed upregulation of genes associated with amino acid biosynthesis ( MET1, MET2, LYS4, TRP4 ), alternative carbon metabolism ( ACS1, GPD2 ), and the TCA cycle ( ACO2, ACC1 ), consistent with nutrient stress in C. albicans . While S. cerevisiae is also known to secrete antifungal metabolites such as phenylethanol and tryptophol 53 , 54 , the inhibitory effects we observed were absent or markedly diminished when direct contact between organisms was prevented. Based on the results of both transcriptomic and contact-dependence assays, we conclude that inhibition is not solely driven by diffusible metabolic products. Rather, contact appears to facilitate localized nutrient depletion. This dependence on contact is a trait of S. cerevisiae previously proven as S. cerevisiae and C. albicans co-aggregate through ALS gene-mediated interactions 55 , imposing physical constraints on C. albicans and intensifying metabolic competition. Additionally, the loss of S. cerevisiae’s inhibitory effects upon heat inactivation suggests that it depends on metabolically active cells. However, as heat inactivation also alters cell surface properties 56 , the observed C. albicans transcriptional reprogramming may result from both metabolic competition and altered cell-cell interactions, warranting further investigation. Building on this, transcriptomic profiling revealed that S. cerevisiae interferes with C. albicans biofilm formation on epithelial cells through both physical disruption (downregulation of TRY5 , SUC1 , SAP8, HGC1, YAK1 ) and metabolic interference (downregulation of HGT13, GAL1, MAL31 ). We found that S. cerevisiae integrated into C. albicans biofilms on epithelial cells, reducing biofilm density and displacing C. albicans cells toward the vaginal lumen, where clearance is likely enhanced 57 , 58 . This resembles Lacticaseibacillus rhamnosus –mediated displacement of C. albicans from intestinal epithelia 59 . The downregulation of adhesion, morphogenesis, and biofilm genes in C. albicans suggests structural repression, likely driven by S. cerevisiae -induced metabolic competition. This supports a dual mechanism where metabolic and physical interference jointly disrupt biofilm integrity. While this trait has been reported in other contexts, it is novel in VVC. By disrupting biofilm structural integrity, S. cerevisiae diminishes the biofilm’s protective properties against treatment and immune cell infiltration 60 , 61 . This weakening may enhance the efficacy of conventional antifungal therapies, providing a potential avenue for synergistic treatment strategies 62 . Collectively, the multifaceted inhibitory mechanisms of S. cerevisiae directly against C. albicans already position it as a promising therapeutic candidate for C. albicans -induced VVC. While C. albicans virulence is a key factor in VVC pathology, a dysregulated inflammatory response drives symptom severity 8 , 63 . This is supported by elevated activation of key inflammatory signaling pathways (NF-κB, c-Fos, and p38) in both symptomatic and asymptomatic women colonized with hyphae-forming C. albicans strains 8 , 64 . We observed that reductions in C. albicans virulence by Sc3458 were accompanied by dampened epithelial immune responses. The reduced C. albicans colonization and hyphal morphology were associated with reduced activation of MAPK and NF-κB pathways, both critical for driving pro-inflammatory cytokine production in VECs 30 . Notably, S. cerevisiae has been shown to modulate these same pathways in other host environments 25 , suggesting a conserved mechanism of immune modulation. Within the NF-κB pathway, reduced expression of key components such as RELA (p65), RELB , and NEMO was observed, potentially impairing pro-inflammatory cytokine transcription 30 , 65 . Similarly, downregulation of MAPK components, including MKK7 and MLK3 , suggests diminished JNK and p38 pathway activation 66 , 67 , while reduced ERK1 expression, associated with cell proliferation and stress responses, indicates decreased MAPK signaling overall. This reduction in MAPK signaling could decrease pro-inflammatory cytokine production, potentially mitigating mucosal inflammation during VVC 37 , 65 . Furthermore, S. cerevisiae presence downregulated PI3K/Akt pathway components, including AKT1 , AKT2 , and IRS1 , further supporting an overall dampening of epithelial inflammatory signaling. Downregulation of pattern recognition and growth factor receptors such as TLR2 and FGFR1/4 , may further contribute to limiting immune activation and epithelial stress responses 34 . While transcriptional changes may not directly reflect protein activity, the transcriptional shifts to a less inflammatory state align well with reduced fungal burden, inhibition of the yeast-to-hypha transition, decreased biofilm formation and inflammatory cytokine release from VECs. This scenario likely lowers IL-1 axis cytokines that drive uncontrolled neutrophil activation in VVC 8 . Correspondingly, we observed reduced epithelial damage alongside decreased IL-1α release. Similarly, monocyte-derived macrophages also showed reduced IL-1β responses upon Sc3458 presence. Given that these responses are critically dependent on the C. albicans toxin candidalysin 41 , 68 – 70 , it is plausible that S. cerevisiae interferes with toxin delivery, thereby mitigating inflammation and cell death 28 . Importantly, in vivo Sc3458 treatment similarly suppressed IL-1 responses, while neutrophil chemoattractants such as CXCL1 and CXCL5 remained present or elevated. Despite slightly reduced neutrophil numbers, fungal burden was significantly lowered, suggesting that Sc3458 enhances neutrophil efficacy while mitigating IL-1-driven hyperinflammation, a hallmark of dysfunctional immunity in VVC 8 . VVC is suggested to involve type 2 immunity, with IL-4 implicated in the mucosal allergy-like response seen in severe cases 71 . Our data demonstrate that Sc3458 markedly reduced the levels of both IL-4 and CCL11 in vaginal lavage fluid, alongside a reduction in IL-31, a cytokine associated with pruritus. Together, these findings point to Sc3458's potential to also alleviate symptomatic infection by moderating allergic inflammation. On top of that, while innate immunity is the primary defense against C. albicans in the vagina, the extended in vivo timeline allowed us to assess adaptive responses. Sc3458 treatment led to a marked reduction in IL-17A and IL-22 levels, indicating suppression of Th17-mediated inflammation 8 . Although IL-17 and IL-22 are not strictly required for fungal control in all VVC models, their overactivation can drive mucosal inflammation. Notably, controlled elevation of IL-22 has been linked to protection through antimicrobial peptide induction 72 . Thus, Sc3458 may fine-tune Th17 responses, limiting immunopathology while maintaining effective fungal clearance. In contrast, the industrial control strain exacerbated inflammation, inducing higher levels of IL-1α, IL-1β, and IL-4. This indicates that IC promotes both hyperinflammation and mucosal allergy responses, which could aggravate symptoms. Although C. albicans load and epithelial signaling are reduced by S. cerevisiae , treatment is unlikely to begin immediately at infection onset, allowing time for dysregulated non-self-limiting neutrophil-mediated inflammation 8 . Consequently, S. cerevisiae’ s effect on VVC pathology may remain limited without directly influencing neutrophil activation and efficacy. We observed reduced epithelial-mediated neutrophil activation in the presence of S. cerevisiae in vitro , including lower IL-8 and ROS levels, which are associated with heightened inflammation and exacerbated VVC symptoms 71 , 73 . Neutrophil surface marker expression suggested an altered activation state. Downregulation of CD35 and CD66b suggested reduced degranulation and release of tissue-damaging enzymes in response to C. albicans -infected VECs 74 . In contrast, CD62L, typically shed during activation, was preserved, indicating a less activated neutrophil phenotype 75 . Conversely, the chemokine receptor CXCR2 which is internalized upon chemokine sensing, showed moderate downregulation, supporting recent or active neutrophil recruitment 76 . While CD16, linked to antimicrobial functionality, was upregulated 77 . Together, these in vitro findings highlight that S. cerevisiae tempers alters the neutrophil activation state while preserving the ability to effectively counteract the infection. Importantly, CD11b—an integrin involved in neutrophil adhesion, recruitment, and recognition of C. albicans antigens such as Pra1 and β-glucan 78 , 79 —was also decreased, pointing to diminished activation potential by direct fungal recognition. While S. cerevisiae had no striking effects on macrophage and granulocyte infiltration in vivo , the activation state of eosinophils, particularly their elevated integrin expression, was congruent with human neutrophils, decreased in the presence of S. cerevisiae . Beyond being highly upregulated on eosinophils by fungal exposure 42 , 80 , CD11b/c are both antifungal recognition receptors 42 , 81 , and eosinophils heavily rely on CD11b for release of allergy-promoting molecules when engaging fungi 42 , 82 , 83 . This indicates that S. cerevisiae treatment can moderate granulocytic inflammation of both neutrophils and eosinophils. Despite dampening granulocyte activation, surprisingly, the treatment with S. cerevisiae significantly enhanced neutrophil killing of C. albicans and suppressed its overgrowth, without inducing excessive NET formation or compromising neutrophil viability. That S. cerevisiae facilitates better killing, while even dampening neutrophil ROS, argues for a more directed antifungal response and less collateral damage 84 . Enhanced C. albicans clearance may stem from improved recognition of the fungus, enabling more effective neutrophil responses, such as precise NET formation or enhanced phagocytosis. While C. albicans masks β-glucans with mannoproteins to evade detection, S. cerevisiae exposes β-glucans, activating Dectin-1-mediated neutrophil responses and countering immune evasion, enhancing neutrophil phagocytosis, oxidative burst, and antifungal activity 85 – 88 . Although it remains unclear whether S. cerevisiae itself is phagocytosed to a significant extent, it does not appear to act as a harmful distraction for immune cells. Instead, its exposed β-glucans may prime or potentiate antifungal responses, thereby contributing to enhanced C. albicans clearance. Additionally, C. albicans metabolic stress in the presence of S. cerevisiae may increase vulnerability to oxidative stress through impairing its ability to detoxify ROS 89 . Further, by inhibiting filamentation, S. cerevisiae enables neutrophils to prioritize efficient antimicrobial mechanisms like phagocytosis and intracellular killing over tissue-damaging degranulation and oxidative burst 90 , 91 . Additionally, S. cerevisiae treatment enhanced neutrophil-mediated C. albicans biofilm clearance. While the underlying mechanism remains to be fully elucidated, it may involve disruption of biofilm-associated barriers—including extracellular matrix density, hyphal architecture, and altered PAMP expression—that typically impair immune recognition and contribute to fungal persistence 92 . As previously highlighted, enhanced neutrophil efficacy was also evident in vivo : despite slightly reduced neutrophil numbers and a dampened IL-1–driven inflammatory environment, Sc3458 treatment led to a significant reduction in C. albicans burden. This supports the notion that Sc3458 not only modulates neutrophil activation but also enhances their antifungal function in a physiologically relevant setting. Interestingly, while the industrial control strain also improved neutrophil-mediated C. albicans killing in vitro , our in vivo findings revealed only transient antifungal effects, accompanied by a pronounced pro-inflammatory and allergic response. These contrasting outcomes underscore the strain-specific immunomodulatory properties of S. cerevisiae . Additionally, given the physiological and microbiome differences between mice and humans, further studies, particularly clinical studies, are needed to validate these findings and elucidate the underlying pathways driving neutrophil modulation. Our broad screening of S. cerevisiae isolates sets Sc3458 apart from other strains as a uniquely effective candidate therapeutic agent. We highlight the potential of Sc3458 as a dual-action VVC treatment, uniquely targeting both C. albicans pathogenicity and modulating immune responses (Fig. 8 ). By reducing fungal load, S. cerevisiae alleviates epithelial stimulation, specifically the pro-inflammatory IL-1 axis and prevents excessive neutrophil activation, thereby mitigating inflammation and epithelial damage. Simultaneously, it recalibrates neutrophil antifungal mechanisms, enhancing fungal clearance without reliance on tissue-damaging responses such as excessive ROS production. Collectively, this translated to improved fungal clearance and a shift in the immune response away from IL-1-driven and allergic hyperinflammation upon treatment of mice with Sc3458. These findings not only advance our understanding of fungal-host-pathogen interactions but also pave the way for clinical studies to validate S. cerevisiae as a live biotherapeutic agent to manage fungal infection and inflammatory responses in VVC. Material and methods C. albicans and S. cerevisiae strains and culture conditions. During this research, the strains utilized included C. albicans SC5314 101 , CA3153 102 , and I49 (vaginal isolate). For the S. cerevisiae strains, various S. cerevisiae strains were employed, namely 4609 (PYCC), 6883 (DBVPG), 6886 (DBVPG), 6888 (DBVPG), 6894 (DBVPG), 3458 (NCYC), 3471 (NCYC), 3076 (NCYC), Y.00240 (NCAIM), S288C (Cold Spring Harbor Laboratory), and CNCM I-3856 (Cerevisia®). Before conducting all assays, individual colonies were selected from yeast extract peptone dextrose (YPD) agar plates and transferred to liquid YPD medium. Subsequently, the cultures were incubated overnight in a shaking incubator (240 rpm) at 30°C. The cells were harvested by centrifugation (4500 rpm, 1 min), subjected to two washes in 1 x phosphate-buffered saline (PBS), and adjusted to the desired optical density. Growth evaluation. Growth was assessed by adjusting the optical densities of C. albicans and S. cerevisiae cells to 0.1 and 0.5 at 600 nm, respectively. The evaluation was conducted in VSM at a pH of 4.2 (Table S2) or in RPMI 1640 medium (Gibco) 103 , 104 . C. albicans growth in the co-cultures was assessed at 37°C and 760 rpm in 96-well plates, after 6 and 24 hours, using fluorescently tagged C. albicans strains. Fluorescence measurements (Excitation: 569nm, Emission: 600nm) of C. albicans , tagged using the Cip10-mScarlet-IDT plasmid 105 (CaSC5314 ACT1p -mScarlet, CaCA3153A ACT1p -mScarlet, CaI49 ACT1p -mScarlet), were performed using a Synergy™ H1 microplate reader (BioTek). Imaging of cultures. The imaging of growth was conducted according to the protocol used for the growth evaluation assay. Cocultures were visualized using a Fluoview FV1000 confocal laser scanning microscope, with the GFP fluorophore excited by a 488 nm laser and emission detected through a BA505–565 bandpass filter 105 . Five representative, randomly selected positions were imaged for each condition, and a representative image was selected. Culturing of vaginal epithelial cells. To effectively mimic the vaginal epithelial layer, A-431 VECs (ACC 91) were employed. These cells, derived from vulva epidermoid carcinoma, are frequently utilized as a representative model for the vaginal niche 106 , 107 . Before experimentation, the cells were screened for mycoplasma contamination using a PCR mycoplasma test kit (Sigma Aldrich). A-431 VECs were cultured in RPMI 1640 medium (Gibco), supplemented with 10% fetal calf serum (FCS), and maintained at 37°C in a 5% CO2 atmosphere with constant humidity. During experimental assays, cells were seeded at the desired density. Adhesion to VECs. VECs were seeded at a density of 2 x 10 4 cells per well in 96-well plates and incubated at 37°C with 5% CO2 for two days, or until confluent. C. albicans and S. cerevisiae cells were both adjusted to a Multiplicity of Infection (MOI) of 5. VECs were infected with C. albicans cells, with or without an S. cerevisiae strain (100 µL of 10 6 cells/mL in RPMI 1640 without FCS), and incubated for 90 min at 37°C and 5% CO2. In the assessment of S. cerevisiae's potential to displace C. albicans , C. albicans was initially introduced alone for 90 min. Subsequently, non-adherent cells were removed by rinsing with PBS, and the S. cerevisiae cells were added for an additional 90 min. For the final readout, the medium was removed, and the wells were gently rinsed with PBS to eliminate non-adherent or loosely adherent cells. To detach the adhered C. albicans cells, the wells underwent trypsinization using 0.5% trypsin EDTA (Gibco), followed by sonication and dilution for a suitable colony forming units (CFU) readout on CHROMagar™ Candida (BioTrading), enabling differentiation between C. albicans and S. cerevisiae cells. Filamentation evaluation. C. albicans filament formation was assessed in both VSM and RPMI. The experimental setup was similar to that of the growth assay, except for adjusting the VSM pH to 5 to facilitate early filament formation. Following a 3-hour incubation at 37°C at 400 rpm, filament formation was quantified by manually counting filamentous cells using a Burker chamber under a Zeiss microscope. Epithelial damage to VECs. VECs were seeded as described in the adhesion assay and infected with C. albicans and S. cerevisiae cells as described above, however, both at MOI 0.5, and incubated for 24 hours at 37°C with 5% CO2. To assess necrotic epithelial damage, the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) was measured using the Cytotoxicity Detection Kit from Roche 108 . LDH activity was quantified in the supernatant of infected VEC monolayers in 96-well plates 24 hpi. LDH from rabbit muscle (5 mg/ml, Roche) was utilized to establish a standard curve for determining LDH concentrations. The background LDH activity from uninfected VECs was subtracted from the test conditions. Cytokine release by VECs. Similar to the setup above, supernatants from VECs infected with C. albicans , with or without the addition of S. cerevisiae , were analyzed for the presence of inflammatory cytokines (IL-α, IL-8, GM-CSF). Supernatant cytokine concentrations were measured using human enzyme-linked immunosorbent assays (ELISA; R&D Systems) following the manufacturer’s instructions. Cluster analysis. Cluster analysis was performed to identify patterns of similarity and the top-performing S. cerevisiae strains based on their rankings across the assays. Clustering quality was assessed using the silhouette width metric, which ranges from 1 (indicating a strong fit) to -1 (indicating a poor fit). To determine the optimal number of clusters (k), K-means and K-medoids algorithms were applied, with the best clustering identified by maximizing the silhouette width 109 . Data analysis and visualization were performed using RStudio (RStudio Team, 2020) with the R programming language. RNA isolation and pooling. VEC were seeded in 6-well plates at 3 × 10 5 cells per well and cultured until confluent for 2 days. Following medium removal, VECs were infected with C. albicans at MOI 5, either alone or with S. cerevisiae at MOI 5 (750 µL of 3 x 10 6 cells/mL in RPMI 1640 without FCS) at 37°C with 5% CO2. RNA samples were collected at 6 and 24 hpi. Plates were centrifuged cool for 5 min at 2000 rpm, after which the supernatant was replaced with 500 µL of RLT buffer (QIAGEN), containing 1% β-mercaptoethanol. The cells were detached using a cell scraper, immediately shock-frozen in liquid nitrogen, and stored at -80°C until further use. For RNA extraction, the samples were thawed on ice and centrifuged for 10 min at 14000 rpm at 4°C. The supernatant was collected and transferred to a new microcentrifuge tube for the isolation of human RNA, while the resultant pellet was used to isolate fungal RNA. Both human and fungal RNA were isolated using the TRIzol-chloroform method, as previously described 110 . The RNA concentration and quality of both human and fungal RNA were assessed using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). To ensure sufficient library depth for both human and fungal RNA, we evaluated various pooling strategies 111 . Based on these evaluations, fungal and human RNA samples were combined in a 1:2 ratio by concentration for library preparation and sequencing, aiming to achieve adequate sequencing depth for comprehensive differential gene expression analysis. Before pooling, we measured cross-mapping rates by aligning reads from strictly human and fungal samples to respective fungal and human genomes, resulting in no cross-mapped reads between the three species. RNA-seq library preparation and sequencing. Bulk RNA sequencing (RNA-seq) libraries were prepared by Novogene (Directional mRNA enrichment libraries) and subjected to NovaSeq paired-end sequencing (2 × 150 base pairs). Sequencing depth and read length were optimized to ensure comprehensive coverage and reliable differential gene expression analysis. RNA-seq pipeline. Paired-end FASTQ files were obtained from Novogene for subsequent analysis. Initial quality control of the raw sequencing data was conducted using FastQC and MultiQC 112 , 113 . For read alignment and quantification, we employed the splice junction-sensitive aligner STAR 114 . Given that the samples included RNA from both host and pathogen, we performed read mapping against a concatenated reference comprising both the human and C. albicans genomes. Human reads were aligned to the primary genome assembly GRCh38, with annotations derived from NCBI (last accessed May 2, 2024). The C. albicans SC5314 genome assembly ASM18296v3 and associated annotations were sourced from NCBI (last accessed May 2, 2024). BEDtools (intersectBed) and Picard were used to remove rRNA reads and optical duplicates from the mapped reads 115 , 116 . Samtools was used for indexing and generating summary statistics 117 . htseq-count was used to count the number of genes within each BAM file 118 . Coverage was evaluated using deepTools (bamCoverage) and BEDtools 116 , 119 . Sequence alignment was assessed using BLAST 120 . Data analysis and visualization were performed using RStudio (RStudio Team, 2020) with the R programming language. Differential gene expression analysis was performed using the Bioconductor package edgeR v.3.34.0 121 . Read counts were obtained from STAR mapping. For human samples and each fungal species, we compared time point 0 with other time points throughout the infection course using the glmQLFTest function from edgeR. We used filterByExpr to filter out low-count genes. The dispersions were estimated using the estimateDisp function, and differential expression was assessed by applying a contrast matrix with glmQLFTest. Genes with a log2(fold change) > 1.0 and an adjusted P (FDR) < 0.05 were considered differentially expressed. To visualize the data, we performed Principal Component Analysis (PCA) on normalized counts and created plots using ggbiplot. Differentially expressed gene results were visualized with scatter and volcano plots. Gene ontology enrichment was performed using ShinyGO 122 . Following this, the significantly enriched GO terms were analyzed using REVIGO85, applying a similarity threshold of Small (0.5) 123 . All code and scripts written for this paper can be accessed on the following online repository: https://github.com/dania-ab/VVC Biofilm biomass. C. albicans biomass formation on VECs was assessed by measuring the fluorescence emitted by fluorescently tagged C. albicans strains. The experimental procedure followed the adhesion assay protocol: VECs were seeded and cultured until confluent, then infected with C. albicans , with or without S. cerevisiae . At 24 and 48 hpi, the medium was aspirated, and the wells were gently washed with PBS to remove non-adherent or loosely attached cells. Subsequently, biomass quantification was performed using fluorescence measurements on a Synergy™ H1 microplate reader (BioTek), with excitation set to 569 nm and emission to 590 nm 105 . For experiments involving transwells, the cells were physically separated using a ThinCert™ Cell Culture insert (Greiner Bio-one) with a 0.4 µm pore size. For experiments involving heat-inactivated S. cerevisiae cells, inactivation was performed by incubating the cells in 1X PBS at 65°C for 30 minutes. Biofilm distribution. Biofilms were established using the same protocol as the biofilm biomass assay. At 24 hpi, the supernatant was collected, and the wells were gently washed with PBS to remove non-adherent or loosely attached cells. To detach the adhered C. albicans cells, the wells were treated with 0.5% trypsin-EDTA (Gibco), followed by water bath sonication. The resulting cell suspension was then diluted to obtain an appropriate CFU count on CHROMagar™ Candida (BioTrading). Biofilm structure. Biofilms were established following the same protocol as the biofilm biomass assay but were grown on IBIDI plates. Imaging of the biofilms was performed using a Fluoview FV1000 confocal laser scanning microscope. The mScarlet fluorophore was excited with a 559 nm laser, and emission was captured through a BA575–675 bandpass filter 105 . A series of images were acquired at 4.51 µm intervals along the z-axis, allowing for a three-dimensional reconstruction of the biofilms from the start to the end of the fluorescent signal. The z-stack images were subsequently exported to the Napari interface for further analysis 124 . Preparation of hMDMs. Buffy coats obtained from healthy volunteers underwent Histopaque-1077 (Sigma-Aldrich) density centrifugation to isolate human peripheral blood mononuclear cells (hPBMC). CD14-positive monocytes were subsequently isolated using magnetic automated cell sorting (autoMACs; MiltenyiBiotec). These cells were then differentiated into human monocyte-derived macrophages (hMDMs) by seeding 1.7 x 10 7 cells in a 175 cm 2 cell culture flask containing RPMI 1640 medium with 2 mM l -glutamine (Thermo Fisher Scientific), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Bio&SELL), and 50 ng/mL recombinant human M-CSF (ImmunoTools). The cells were incubated for seven days at 37°C and 5% CO2 with a medium exchange on day 5. The adherent hMDMs were detached using 50 mM EDTA in PBS, then seeded in 96-well plates at a final concentration of 4 x 10 4 hMDMs/well in RPMI + FBS + M-CSF, and incubated overnight. The macrophage infection experiments were conducted in serum-free RPMI medium. hMDMs stimulation. To assess the stimulation of hMDMs, the release of IL-1β was measured, as it serves as an indicator of NLRP3 activation 125 . hMDMs were seeded at a density of 4 × 10 4 cells per well, as previously described. Before infection, the phagocytes were primed by incubating them with 50 ng/mL of lipopolysaccharide (LPS) (Sigma Aldrich) for 2 hours. Subsequently, the hMDMs were infected with C. albicans at MOI 0.5, with or without S. cerevisiae at MOI 0.5, for 24 hours at 37°C with 5% CO 2 . After the designated incubation period, the supernatant from the infected hMDMs was collected by centrifuging the plates at 250 x g for 5 min. Supernatant IL-1β concentrations were measured using human enzyme-linked immunosorbent assays (ELISA; R&D Systems) following the manufacturer’s instructions. Isolation of human neutrophils. The isolation of primary human neutrophils followed the procedures outlined by Gresnigt et al. 126 . Initially, human peripheral blood mononuclear cells were separated from granulocytes and erythrocytes using density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich) in a sterile 50 mL falcon. Neutrophils were subsequently extracted from the erythrocyte/granulocyte fraction by hypotonic lysis with a buffer containing 155 mM NH 4 Cl and 10 mM KHCO 3 . Following two washes in 1 x PBS, the neutrophils were resuspended in RPMI medium and adapted to the required concentration. Neutrophil stimulation: IL-8 release. To assess neutrophil stimulation in response to C. albicans and the potential influence of S. cerevisiae , neutrophils were treated with supernatants. These supernatants were diluted 1:2 in RPMI and collected from VECs exposed to C. albicans , with or without S. cerevisiae , for 24 hours. This process was carried out as previously described. Freshly isolated neutrophils were seeded at 1 × 10 5 neutrophils/well in a 96-well plate and following a 3-hour stimulation period at 37°C with 5% CO 2 , supernatants from the neutrophils were collected. The concentration of IL-8, an indicator of neutrophil activation, was then measured in these supernatants using an ELISA assay (R&D Systems) following the manufacturer’s instructions 127 . Neutrophil activation: ROS production. Neutrophil activation in response to C. albicans , with and without S. cerevisiae , was evaluated using ROS production in an oxidative burst assay. To quantify the total ROS, a luminol-enhanced chemiluminescence method was performed. Freshly isolated neutrophils were seeded at 1 × 10 5 neutrophils/well as mentioned above. Neutrophils were then incubated for 30 min at 37°C and 5% CO 2 , to let them attach. Subsequently, the neutrophils were stimulated for 1 hour 37°C and 5% CO 2 with undiluted supernatants from VECs exposed to C. albicans , with and without S. cerevisiae following the previously described protocol. After incubation, 50 µL of zymosan (at a concentration of 150 µg/mL) was added to further stimulate the fungal immune response and enhance ROS production 128 . Following stimulation, 50 µl of RPMI1640 containing 200 µM luminol (Sigma Aldrich) and 16 U HRP (Sigma Aldrich) for total ROS detection was added. Chemiluminescence was measured every 2.5 minutes for 2.5 hours using a Tecan Infinite M200 microplate reader. The area under the curve was calculated using GraphPad Prism 10.4.3. Neutrophil stimulation: Activation-associated cell surface markers. Further assessment of neutrophil stimulation involved analyzing the regulation of neutrophil activation markers using flow cytometry. The selection of activation markers was based on granulocyte responses to fungal pathogens or related stimuli 41 , 42 . Neutrophils were seeded in a round-bottom 96-well plate at a density of 2 × 10 5 cells per well and stimulated with undiluted supernatants from VECs exposed 24 h to C. albicans , with and without S. cerevisiae , following the previously described protocol. After a 3 h stimulation period at 37°C with 5% CO 2 , supernatants were removed, and the neutrophils were washed with flow cytometry buffer (PBS, 2% FCS). To minimize nonspecific staining, neutrophils were first incubated with Fc-block Human TruStain FcX (BioLegend). Subsequently, a mixture of fluorophore-linked antibodies targeting surface molecules indicative of activation status was added: CD15-APC-Fire750 (W6D3), CD11b-BV421 (ICR44), CD16-PerCP-Cy5.5 (3G8), CD35-FITC (E11), CD62L-AlexaFluor647 (DREG-56), CD66b-PE (G10F5), and CXCR2 (5E8, all from BioLegend). Fixable Viability Dye eFluor506 (Invitrogen) was added to exclude non-viable cells for 20 min at 8°C. Subsequently, cells were washed with flow cytometry buffer, filtered through a 70 µm mesh, and then analyzed using a FACSVerse Cell Analyzer flow cytometer (BD Biosciences). Gating strategies for granulocyte populations and activation markers followed the approach described by Valentine et al. 41 . Data analysis was carried out using FlowJo v.10. Neutrophil chemotaxis. Neutrophil chemotaxis was performed as described by Valentine et al. 41 . Briefly, primary human neutrophils were stained with Cytopainter Green (2 µL in 1 × 10⁶ neutrophils suspended in RPMI), incubated for 10 min at room temperature in the dark. Cells were then washed with Hank’s Balanced Salt Solution containing 20 mM HEPES (pH 7), centrifuged (10 min, 300 × g), and resuspended in endothelial cell medium (ECM, PromoCell) at 5 × 10⁵ cells/mL. Human umbilical vein endothelial cells (HUVECs) were cultured for 72 h in 150-cm² flasks, harvested, and seeded into 3 µm transwell inserts at 2 × 10⁴ cells/insert. After 48 h, inserts were transferred to 24-well plates containing confluent VECs, seeded two days earlier at 1 × 10⁵ cells/well. Medium in the inserts was refreshed (200 µL), and VECs were infected with C. albicans (1 × 10⁵ cells, MOI 0.5) in the presence or absence of S. cerevisiae (1 × 10⁵ cells, MOI 0.5) in a total volume of 600 µL. After 18 h, 200 µL of stained neutrophils were added to the inserts.. Plates were incubated for 2 h at 37°C and 5% CO₂. Images were acquired using an IncuCyte SX5 live-cell imaging system (Sartorius). Thresholds for fluorescence intensity and area size ensured accurate segmentation and quantification. Image analysis was performed using Incucyte 2023A Rev2 software. Neutrophil death and NETosis evaluation. To assess the influence of S. cerevisiae on C. albicans -induced neutrophil responses, co-cultures were established to evaluate NETosis and neutrophil cell death. Neutrophils (1 × 10⁵ cells per well) were pre-stained with Sytox Green (4 × 10⁻⁵ µL/mL RPMI) to monitor NETosis and cell death, while S. cerevisiae and C. albicans were added at an MOI of 0.5. Staining was performed in the dark at 37°C with 5% CO₂ for 15 min prior to co-culture. Co-cultures were imaged in an Incucyte SX5 (Sartorius) at 37°C with 5% CO₂. Negative controls were left unstimulated, positive controls were stimulated with 20 nM PMA. NETosis and cell death were monitored in real-time using green fluorescence imaging. Images were captured from two regions per well every 20–30 min for 12 h at ×20 magnification, with an exposure time of 100 ms. Representative images and images of unstimulated and PMA-induced NETosis conditions were used to train the Incucyte Basic Software for analysis. The Top-Hat method was applied for background correction, and the edge-split tool was used to distinguish closely spaced objects. Thresholds for fluorescence intensity and area size ensured accurate segmentation and quantification. Image analysis was performed using Incucyte 2023A Rev2 software. Neutrophil-mediated C. albicans killing and growth. To evaluate the effects of S. cerevisiae on neutrophil-mediated C. albicans growth impairment, we used a setup similar to that described above, incorporating mScarlet-tagged C. albicans . Fungal overgrowth was monitored in the orange fluorescence channel using the Incucyte live-cell imaging system, with an exposure time of 300 ms. The Adaptive method was applied for image analysis to account for variations in fluorescence intensity and object morphology during fungal growth. Data were analyzed using Incucyte 2023A Rev2 software. To assess the influence of S. cerevisiae on neutrophil-mediated C. albicans killing, a similar co-culture system was used, except that unstained neutrophils and C. albicans were included. Killing assays were performed at 1 and 3 hpi. After co-culture, neutrophils were lysed with 4% Triton X-100, and samples were scraped, sonicated, serially diluted, and plated on CHROMagar™ Candida (BioTrading) to differentiate C. albicans from S. cerevisiae . Neutrophil-mediated C. albicans biofilm eradication. To evaluate whether the biofilm-inhibiting effects of S. cerevisiae enhance C. albicans clearance by neutrophils, we assessed neutrophil-mediated killing in co-culture with C. albicans biofilms. Following the biofilm biomass quantification protocol, co-cultures were incubated for 24 h, washed to remove non-adherent cells, and fluorescent C. albicans values were measured to normalize the data, accounting for reduced biofilm biomass due to S. cerevisiae . 5 × 10⁵ neutrophils per well were added and incubated with the biofilms for 3 hours at 37°C with 5% CO₂ to allow for C. albicans killing. After co-culture, neutrophils were lysed with 4% Triton X-100, and biofilms were scraped, sonicated, serially diluted, and plated on CHROMagar™ Candida (BioTrading) to differentiate C. albicans colonies from S. cerevisiae . This method enabled precise quantification of C. albicans survival and biofilm clearance. Murine model of VVC. Female BALB/cJ mice 8 weeks old were obtained from Charles River and used for the study. To induce a pseudoestrous state, mice received a subcutaneous injection of 0.1 mg β-estradiol 17-valerate (Sigma), dissolved in 100 µL of sesame oil, 72 hours before inoculation. Estrogen administration was continued weekly throughout the experiment 129 . To establish infection, estrogen-treated mice were sedated and inoculated with 10 µL of 10 7 bioluminescent (BLI) C. albicans cells/mL, suspended in 1 × PBS. The inoculum was administered intravaginally via pipetting. To minimize leakage and enhance fungal retention, mice were positioned with their hindquarters elevated for 20 minutes post-inoculation. Following a 24-hour incubation period to allow for infection establishment, mice received intravaginal treatment with either 10 µL of 10 8 S. cerevisiae 3458 cells/mL or 1 × PBS (control), similar to C. albicans infection. Treatments were initiated one day post-inoculation and continued daily. The experimental protocols were reviewed and approved by the Ethical Committee for Animal Experimentation of KU Leuven under approval number Bio-157/2024. Monitoring C. albicans vaginal load. Fungal burden was assessed on days 2, 4, 6, 8, and 10 post-infection using bioluminescence imaging (BLI). Mice were anesthetized with 2.5–3.5% (v/v) isoflurane gas, followed by maintenance at 1.5–2% isoflurane in oxygen (Piramal Critical Care). D-luciferin (1.1665 mg/mL, 10 µL) was administered intravaginally. After anesthesia induction, mice were positioned supine in an IVIS Spectrum System (Perkin-Elmer, Hopkinton, MA, USA). Imaging began 5 minutes post-injection, capturing five consecutive images using the following parameters: 30-second exposure time, medium binning, F/stop of 1, and subject height of 1.5 cm. Bioluminescence signal quantification was performed using Living Image Software (version 4.7.3, Perkin-Elmer). Total photon flux (photons/second) was measured within a region of interest (ROI) of 2.8 cm 2 , covering the vaginal area. The maximum signal from consecutive acquisitions was used for further analysis. Monitoring inflammatory state. To assess the presence of inflammatory markers, vaginal lavages were performed. Each lavage was conducted using 200 µL of 1 × PBS, administered in four consecutive volumes of 50 µL each. Following collection, the lavage samples were centrifuged at 1200 rpm for 10 minutes at 4°C to separate soluble components from immune cells. The resulting supernatants were collected and subjected to quantitative analysis using a Luminex Mouse Discovery Assay. Flow cytometry. Flow cytometry was performed to assess the presence of macrophages, neutrophils and eosinophils in vaginal lavage fluid. Lavage samples were centrifuged at 1,200 rpm for 10 min at 4°C to separate soluble components and immune cells. The resulting cell pellet was washed with ice-cold 1 x PBS and incubated with Fc-receptor blocking antibodies (anti-CD16/CD32 Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 minutes at room temperature in the dark ( Table S3 ). Simultaneously, a Zombie Aqua™ viability dye (BioLegend, USA) was applied to discriminate live from dead cells. Following incubation, the cells were washed with cold 1 x PBS supplemented with 2% FCS-2mM EDTA and stained with a surface marker antibody cocktail ( Table S3 ) at 4°C for 30 minutes. After staining, cells were washed with cold PBS containing 2% FBS and resuspended in PBS supplemented with 2% v/v FBS and 2 mM EDTA to prevent cell aggregation. Samples were maintained at 4°C until analysis. Flow cytometry was conducted using a BD LSRFortessa™ X-20 (BD Biosciences) and data analysis was carried out using FlowJo software (BD Biosciences, v10). The specific gating strategies are depicted in Fig. S10 . Declarations Acknowledgments We acknowledge Marisa Valentine and Sophie Austermeier for their technical assistance with macrophage and neutrophil functional assays. We also thank Wouter Van Genechten for his contributions to confocal microscopy imaging and the development of custom scripts for quantitative analysis of biofilm thickness. Nele Berghmans is acknowledged for her support with flow cytometry acquisition and analysis. Funding FWO-funded SBO project DeVEnIR S006424N (P.V.D., M.S.G., S.L.) FWO research community on Biofilms (W000921N) (P.V.D.) FWO PhD fellowship 1SD8622N (M.S.) Flanders Innovation & Entrepreneurship VLAIO HBC.2021.824 (S.B.) FWO-funded SBO project DeVEnIR S006424N (P.V., K.O.C., I.P.) Microverse visiting scientist grant (M.S.) Exploration Grant of the Boehringer Ingelheim Foundation (BIS) (M.S.G., A.D.) Free State of Thuringia and European Union – Project-ID 2023 FGI 0004. "A Live broadcast of the interactions between host and fungal pathogens" (M.S.G, A.D.) German Research Foundation (Deutsche Forschungsgemeinschaft - DFG) Emmy Noether Program 434385622 / GR 5617/1-1 (M.S.G.) Rega Foundation (M.G.) Emmy Noether Programme (D.E.M) Author contributions Conceptualization: M.S., P.V.D., M.S.G., L.D., S.L. Methodology: M.S., D.E.A., S.B., D.E.M., G.V.V., A.R.S., P.V., M.S.G., A.D., L.D., M.G. Investigation: M.S., D.E.A., N.V., S.B., K.O.C., O.V.G., I.P., A.D. Software: M.S., D.E.A. Visualization: M.S., D.E.A., M.S.G., A.D. Funding acquisition: M.S., P.V.D., S.L., L.D., M.S.G. Project administration: M.S., P.V.D. Supervision: P.V.D, M.S.G, S.L., L.D. Writing – original draft: M.S., M.S.G., A.D., D.E.A. Writing – Review & Editing: M.S., D.E.A., L.D., P.V., I.P., A.D., S.L., M.S.G., M.G., P.V.D. References Ilkit, M. & Guzel, A. B. The epidemiology, pathogenesis, and diagnosis of vulvovaginal candidosis: a mycological perspective. Critical reviews in microbiology 37 , 250-261, doi:10.3109/1040841X.2011.576332 (2011). Gonçalves, B. et al. 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Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. The Journal of experimental medicine 197 , 1107, doi:10.1084/jem.20021787 (2003). Fidel, P. L., Cutright, J. & Steele, C. Effects of Reproductive Hormones on Experimental Vaginal Candidiasis. Infection and Immunity 68 , 651-657, doi:10.1128/iai.68.2.651-657.2000 (2000). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryMaterials.docx Supplementary materials Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6795973","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":469270010,"identity":"b7ffc49c-5fae-48c5-929d-adc916d8f9ac","order_by":0,"name":"Mart Sillen","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Mart","middleName":"","lastName":"Sillen","suffix":""},{"id":469270011,"identity":"ba8236f6-53a4-4316-932f-6143d804cdf1","order_by":1,"name":"Dania El Abyad","email":"","orcid":"https://orcid.org/0009-0004-2083-0236","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Dania","middleName":"El","lastName":"Abyad","suffix":""},{"id":469270012,"identity":"93a3b3e7-cf47-46d5-b761-aef019b0c7c3","order_by":2,"name":"Nina Vreys","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"","lastName":"Vreys","suffix":""},{"id":469270013,"identity":"c4b675b0-7124-442e-8352-933659be0236","order_by":3,"name":"Silke Baldewijns","email":"","orcid":"https://orcid.org/0000-0003-1763-6868","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Silke","middleName":"","lastName":"Baldewijns","suffix":""},{"id":469270014,"identity":"6af546ec-9d96-44b1-852c-ff723e6f3e3c","order_by":4,"name":"Ilse Palmans","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Ilse","middleName":"","lastName":"Palmans","suffix":""},{"id":469270015,"identity":"a3f66138-44e7-4695-88c4-120c98cab43d","order_by":5,"name":"Odessa Van Goethem","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Odessa","middleName":"Van","lastName":"Goethem","suffix":""},{"id":469270016,"identity":"f73042e2-1a20-4cf7-89f7-853e467e4892","order_by":6,"name":"Kar On Cheng","email":"","orcid":"","institution":"Leibniz Institute for Natural Product Research","correspondingAuthor":false,"prefix":"","firstName":"Kar","middleName":"On","lastName":"Cheng","suffix":""},{"id":469270019,"identity":"c2bafddc-1e5d-44c8-8071-b329a43bb9dd","order_by":7,"name":"Greetje Vande Velde","email":"","orcid":"https://orcid.org/0000-0002-5633-3993","institution":"KU Leuven - University of Leuven","correspondingAuthor":false,"prefix":"","firstName":"Greetje","middleName":"Vande","lastName":"Velde","suffix":""},{"id":469270020,"identity":"a03d98a2-e97c-4b1f-973d-a05f53c31aa8","order_by":8,"name":"Dolly Estella Montaño Espinosa","email":"","orcid":"","institution":"Hans Knöll Institute (HKI)","correspondingAuthor":false,"prefix":"","firstName":"Dolly","middleName":"Estella Montaño","lastName":"Espinosa","suffix":""},{"id":469270017,"identity":"9311c424-1e6c-4fcf-b7ef-100e1927d727","order_by":9,"name":"Mieke Gouwy","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Mieke","middleName":"","lastName":"Gouwy","suffix":""},{"id":469270018,"identity":"66b816d9-aba9-47c1-aea0-9e27288787f5","order_by":10,"name":"Agustin Reséndiz-Sharpe","email":"","orcid":"","institution":"KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Agustin","middleName":"","lastName":"Reséndiz-Sharpe","suffix":""},{"id":469270021,"identity":"fd8730a7-5f2e-4871-85e5-d8e3b1ecc133","order_by":11,"name":"Axel Dietsschmann","email":"","orcid":"","institution":"Hans Knöll Institute (HKI)","correspondingAuthor":false,"prefix":"","firstName":"Axel","middleName":"","lastName":"Dietsschmann","suffix":""},{"id":469270022,"identity":"8bc3ddd4-0289-4e3d-8be1-5c3ec531c5ac","order_by":12,"name":"Paul Vandecruys","email":"","orcid":"","institution":"Laboratory of Molecular Cell Biology, Department of Biology, KU Leuven","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Vandecruys","suffix":""},{"id":469270023,"identity":"f3d2713f-88a6-4a86-8b3f-1381afbbd630","order_by":13,"name":"Sarah Lebeer","email":"","orcid":"https://orcid.org/0000-0002-9400-6918","institution":"University of Antwerp","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Lebeer","suffix":""},{"id":469270024,"identity":"0964d058-bcb4-464e-877d-7b0b1820d66c","order_by":14,"name":"Liesbeth Demuyser","email":"","orcid":"","institution":"KU Leuven Laboratory of Molecular Cell Biology","correspondingAuthor":false,"prefix":"","firstName":"Liesbeth","middleName":"","lastName":"Demuyser","suffix":""},{"id":469270025,"identity":"00ec0961-3fd9-4e0c-8c4d-b7bc25da7f58","order_by":15,"name":"Mark S. Gresnigt","email":"","orcid":"https://orcid.org/0000-0002-9514-4634","institution":"Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"S.","lastName":"Gresnigt","suffix":""},{"id":469270009,"identity":"169a8b43-1066-4523-ae8d-c4cc46252a62","order_by":16,"name":"Patrick Van Dijck","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-1542-897X","institution":"KU Leuven","correspondingAuthor":true,"prefix":"","firstName":"Patrick","middleName":"Van","lastName":"Dijck","suffix":""}],"badges":[],"createdAt":"2025-06-01 13:45:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6795973/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6795973/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84772110,"identity":"47a9492c-ff56-4554-9d2e-5da1de75ecff","added_by":"auto","created_at":"2025-06-17 08:16:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3788727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eisolates inhibit \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e growth, adhesion, and filamentation. A. \u003c/strong\u003eInhibition of \u003cem\u003eC. albicans\u003c/em\u003e (strains SC5314, CA3153, I49) growth by \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates (A–I), laboratory control S288c (LC), and probiotic standard CNCM I-3856 (IC) after 6 hours (upper panel) and 24 hours (lower panel) in VSM. \u003cstrong\u003eB.\u003c/strong\u003e Representative images of fluorescent \u003cem\u003eC. albicans\u003c/em\u003e CA3153 co-cultured with \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates in VSM after 3 hours. Images display growth and filamentation inhibition, with scale bars set at 100 µm (insets, 25 µm). \u003cstrong\u003eC.\u003c/strong\u003e Inhibition of \u003cem\u003eC. albicans\u003c/em\u003e adhesion to VECs by \u003cem\u003eS. cerevisiae\u003c/em\u003e. The upper panel shows competition for initial adhesion, while the lower panel shows displacement of already adhered \u003cem\u003eC. albicans\u003c/em\u003e. \u003cstrong\u003eD.\u003c/strong\u003e Inhibition of \u003cem\u003eC. albicans\u003c/em\u003e filamentation in VSM by \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates after 3 hours.\u003cstrong\u003e \u003c/strong\u003eBeige hyphal and yeast-shaped cells represent \u003cem\u003eC. albicans\u003c/em\u003e, purple cells represent \u003cem\u003eS. cerevisiae\u003c/em\u003e, and pink cells represent VECs. All values represent mean ± standard deviation (s.d.) from n ≥ 3 independent experiments, with individual data points representing the mean of technical replicates. Data were transformed using a Box-Cox transformation to meet linear regression assumptions. Statistical significance was determined using a one-sample t-test against a theoretical mean of 0%, representing \u003cem\u003eC. albicans\u003c/em\u003e alone. Significant differences are indicated as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Schematic overview in panels A-D was created with BioRender.com.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/d31e0dd679b0c80f2b30a4b8.png"},{"id":84772104,"identity":"4e4533ab-7074-4e7b-b644-310ed9d13d11","added_by":"auto","created_at":"2025-06-17 08:16:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2110583,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e isolates modulate epithelial activation and cluster into performance-based groups.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Schematic showing \u003cem\u003eC. albicans\u003c/em\u003e-induced LDH release from damaged epithelial cells, triggering macrophage activation via IL-1α and neutrophil stimulation via IL-8 and GM-CSF. The diagram highlights immune signaling pathways involved in epithelial damage and \u003cem\u003eC. albicans\u003c/em\u003e-driven immune cell stimulation. Beige hyphal cells represent \u003cem\u003eC. albicans\u003c/em\u003e and pink cells represent VECs. \u003cstrong\u003eB.\u003c/strong\u003eThe effect of \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates (A–I), laboratory control (LC, S288c), and probiotic standard (IC, CNCM I-3856) on \u003cem\u003eC. albicans\u003c/em\u003e-induced LDH release by VECs. Data show modulation of LDH levels across \u003cem\u003eC. albicans\u003c/em\u003estrains SC5314, CA3153, and I49. \u003cstrong\u003eC. \u003c/strong\u003eThe effect of \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates on \u003cem\u003eC. albicans\u003c/em\u003e-induced IL-1α release by A-431 VECs. \u003cstrong\u003eD.\u003c/strong\u003e The effect of \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates on \u003cem\u003eC. albicans\u003c/em\u003e-induced IL-8 release by VECs. \u003cstrong\u003eE.\u003c/strong\u003e The effect of \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates on \u003cem\u003eC. albicans\u003c/em\u003e-induced GM-CSF release by VECs. \u003cstrong\u003eF.\u003c/strong\u003e Heatmap ranking \u003cem\u003eS. cerevisiae\u003c/em\u003eisolates based on their performance across \u003cem\u003eC. albicans\u003c/em\u003e virulence inhibition assays. Rankings are presented numerically, with lower numbers indicating stronger inhibitory performance. Two clusters were identified using k-means clustering (k = 2), as shown by the dendrogram on the left. \u003cstrong\u003eG.\u003c/strong\u003e K-means cluster analysis (k = 2) of \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates based on their rankings in \u003cem\u003eC. albicans\u003c/em\u003e virulence assays. Principal component analysis (PCA) shows two distinct clusters (Cluster 1, light blue; Cluster 2, dark blue), with PC1 (50.7%) and PC2 (17.4%) explaining the variance in isolate performance. Heatmap data represent the mean from n ≥ 2 independent experiments. Schematic overview in panel A was created with BioRender.com.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/7f7034ba90216346d1fa8960.png"},{"id":84772103,"identity":"9ecc6ef2-1ad3-4630-bd44-98f2efe9e1d8","added_by":"auto","created_at":"2025-06-17 08:16:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1008614,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;strain 3458 modulates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and epithelial cell transcriptional responses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA,B.\u003c/strong\u003e PCA of transcriptional profiles of \u003cem\u003eC. albicans\u003c/em\u003e (Ca, \u003cstrong\u003eA\u003c/strong\u003e) and vaginal epithelial cells (VECs, \u003cstrong\u003eB\u003c/strong\u003e) at 6 and 24 hpi. The clustering of transcriptional profiles shows the impact of Sc3458 at both time points. \u003cstrong\u003eC.\u003c/strong\u003e Number of DEGs in \u003cem\u003eC. albicans\u003c/em\u003e and VECs at 6 and 24 hpi. The circle size reflects DEG counts, with a notable increase observed at 24 hpi. Genes were selected based on a Log2 fold change \u0026gt; 1 or \u0026lt; −1 and p-value \u0026lt; 0.05. \u003cstrong\u003eD.\u003c/strong\u003e Volcano plot depicting significantly upregulated (red) and downregulated (blue) genes in \u003cem\u003eC. albicans\u003c/em\u003e at 24 hpi in response to treatment with Sc3458. Genes were selected based on a Log2 fold change \u0026gt; 1 or \u0026lt; −1 and p-value \u0026lt; 0.05. \u003cstrong\u003eE,F.\u003c/strong\u003e Gene Ontology (GO) enrichment analysis of downregulated (D, blue) and upregulated (E, red) genes in \u003cem\u003eC.\u0026nbsp;albicans\u0026nbsp;\u003c/em\u003eat 24 hpi. GO terms were identified using ShinyGO (http://bioinformatics.sdstate.edu/go/) and refined via Revigo (similarity threshold of 0.5, http://revigo.irb.hr/). Biological processes are ranked by –Log10(p-value) (Bonferroni corrected), and fold enrichment is visualized by a color gradient. Key processes include biofilm formation, cellular interactions, and metabolic regulation. Data are derived from n = 3 biological replicates.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/6da8e27904a2f3c1b6d8b79a.png"},{"id":84772106,"identity":"3466315c-f648-427a-9f6f-b0d3b0abbba1","added_by":"auto","created_at":"2025-06-17 08:16:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2500851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reduces \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e biofilm biomass, alters gene expression, and disrupts biofilm integrity. A. \u003c/strong\u003eHeatmap of transcriptional changes in biofilm-associated genes of \u003cem\u003eC. albicans\u003c/em\u003eupon exposure to Sc3458. Gene expression is shown as Log2 fold change, with significant downregulation indicated (Log2 \u0026gt; 1 or \u0026lt; −1, p \u0026lt; 0.05). \u003cstrong\u003eB.\u003c/strong\u003e Representative images of mScarlet-tagged \u003cem\u003eC. albicans\u003c/em\u003e biofilms CA3153 on VECs treated with Sc3458 and the industrial control (IC, CNCM I-3856), shown in head and side views. Scale bars = 100 µm. \u003cstrong\u003eC. \u003c/strong\u003eRelative biofilm biomass of \u003cem\u003eC. albicans\u003c/em\u003e strains SC5314, CA3153, and I49 on VECs at early (24 hpi) and mature (48 hpi) stages in the presence of \u003cem\u003eS. cerevisiae\u003c/em\u003e strain 3458 or the industrial control (IC, CNCM I-3856). Biomass is normalized to \u003cem\u003eC. albicans \u003c/em\u003ealone (100%). \u003cstrong\u003eD.\u003c/strong\u003e Biofilm thickness of \u003cem\u003eC. albicans\u003c/em\u003e strains on VECs at 24 hpi, comparing conditions with and without Sc3458 or the industrial control (IC, CNCM I-3856). \u003cstrong\u003eE.\u003c/strong\u003e Redistribution of \u003cem\u003eC. albicans\u003c/em\u003e (SC5314, CA3153, and I49) between biofilm and supernatant fractions during treatment with Sc3458 or with the industrial control (IC, CNCM I-3856).\u003cstrong\u003e F.\u003c/strong\u003e Redistribution of Sc3458 when exposed to \u003cem\u003eC. albicans\u003c/em\u003e (SC5314, CA3153, and I49) biofilm. \u003cstrong\u003eG.\u003c/strong\u003e Relative biofilm biomass of \u003cem\u003eC. albicans\u003c/em\u003e strains with Sc3458 or with the industrial control (IC, CNCM I-3856) in direct contact or physically separated using transwell inserts (0.4 µm pore size) or exposed to heat-inactivated \u003cem\u003eS. cerevisiae\u003c/em\u003e. \u003cstrong\u003eH.\u003c/strong\u003e Schematic model\u003cstrong\u003e \u003c/strong\u003eillustrating the proposed mechanisms by which \u003cem\u003eS. cerevisiae\u003c/em\u003e (purple cells) inhibits \u003cem\u003eC. albicans\u003c/em\u003e biofilm formation (beige cells) on VECs (pink layer). The inhibitory effects include nutrient sequestration, metabolic adaptation by \u003cem\u003eS. cerevisiae\u003c/em\u003eand physical constraints that disrupt \u003cem\u003eC. albicans\u003c/em\u003eadhesion, filamentation, and biofilm structural integrity. All data represent mean ± standard deviation (s.d.) from n ≥ 3 independent experiments, with dots representing the mean of technical replicates. Statistical significance was determined using one-sample t-tests set at 100% (C) or one-way ANOVA with Holm-Šidák post hoc tests to compare treatment conditions within each \u003cem\u003eC. albicans\u003c/em\u003e strain (D–G). Significant differences are indicated as*p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. Schematic overview in panel H was created with BioRender.com.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/60671e56ef0f22ff526f4fa3.png"},{"id":84773097,"identity":"d17df3f7-b024-4ad9-b96a-261614afe1a3","added_by":"auto","created_at":"2025-06-17 08:24:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1399348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;strain 3458 modulates epithelial transcriptional responses and reduces neutrophil activation induced by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A.\u003c/strong\u003e Volcano plot showing significantly upregulated (red) and downregulated (blue) genes in VECs infected with \u003cem\u003eC. albicans\u003c/em\u003e at 24 hpi in the presence of Sc3458. Genes associated with cell death, inflammation, and stress responses are highlighted. B, C. Gene Ontology (GO) enrichment analysis of downregulated (B, blue) and upregulated (C, red) genes in VECs, based on Log2 fold change \u0026gt; 1 or \u0026lt; −1 and p \u0026lt; 0.1. Enriched biological processes were identified via ShinyGO (http://bioinformatics.sdstate.edu/go/) and refined using Revigo (similarity threshold of 0.5, http://revigo.irb.hr/). Terms are ranked by –Log10(p-value), with fold enrichment visualized as a color gradient. Data are from n ≥ 2 biological replicates.\u003cstrong\u003e\u0026nbsp;D.\u003c/strong\u003e IL-1β release by human monocyte-derived macrophages (hMDMs) stimulated with \u003cem\u003eC. albicans\u003c/em\u003e-infected VEC supernatants under the influence of \u003cem\u003eS. cerevisiae\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003eHorizontal lines represent values for the Sc3458-only (blue) condition, the industrial control-only (light blue; IC, CNCM I-3856) negative control (grey), included as a reference. Data are from n ≥ 9 donors. \u003cstrong\u003eE.\u0026nbsp;\u003c/strong\u003eSchematic showing supernatants from \u003cem\u003eC. albicans\u003c/em\u003e–infected VECs (pink), treatment with \u003cem\u003eS. cerevisiae\u003c/em\u003e (purple), collected after 24 h and used to stimulate neutrophils (green) for 3 h. Beige cells represent \u003cem\u003eC. albicans\u003c/em\u003e, illustrating the indirect effect of the treatment on neutrophil responses. \u003cstrong\u003eF.\u0026nbsp;\u003c/strong\u003eIL-8 release by neutrophils stimulated with \u003cem\u003eC. albicans\u003c/em\u003e-infected VEC supernatants in the presence of Sc3458 or the industrial control (IC, CNCM I-3856). Horizontal lines represent values for the Sc3458-only (blue) condition, the industrial control-only (light blue; IC, CNCM I-3856) negative control (grey), included as a reference. Data are from n = 3 donors.\u003cstrong\u003e\u0026nbsp;G, H.\u0026nbsp;\u003c/strong\u003eROS release by human neutrophils stimulated with supernatant from \u003cem\u003eC. albicans\u003c/em\u003e-infected VECs, showing the effects of Sc3458. Horizontal lines represent values for the Sc3458-only (blue) condition, the industrial control-only (light blue; IC, CNCM I-3856) negative control (grey), included as a reference. Data represent n = 5 donors.\u003cstrong\u003e\u0026nbsp;I.\u003c/strong\u003e Heatmap of neutrophil activation markers (CD11b, CD66b, CD62L, CXCR2) after stimulation with supernatant from \u003cem\u003eC. albicans\u003c/em\u003e-infected VECs with or without \u003cem\u003eS. cerevisiae\u003c/em\u003e. Mean fluorescence intensity (MFI) is normalized to unstimulated controls. Data are from n = 5 donors.\u003cstrong\u003e\u0026nbsp;J.\u0026nbsp;\u003c/strong\u003eTranswell migration assay showing neutrophil chemotaxis through an endothelial–epithelial barrier toward \u003cem\u003eC. albicans\u003c/em\u003e–infected VECs with or without Sc3458 or with the industrial control (IC, CNCM I-3856). Data from n = 4 donors. Statistical significance was determined using one-way ANOVA with Holm-Šidák post hoc tests to compare treatment conditions within each \u003cem\u003eC. albicans\u003c/em\u003e strain (D-H,J) and one-way ANOVA with Holm-Šidák post hoc tests to compare the \u003cem\u003eC. albicans\u003c/em\u003e only (Ca only) condition (I). Significant differences are indicated as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Schematic overview in panel E was created with BioRender.com.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/599e5218ab7f80e1765c58de.png"},{"id":84772109,"identity":"9291d1d2-fd5f-4e37-8023-9f8e5ff347ec","added_by":"auto","created_at":"2025-06-17 08:16:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2454944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eneutrophil-mediated killing, biofilm inhibition, and NETosis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in the presence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ecerevisiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. A.\u003c/strong\u003e Neutrophil-mediated killing of \u003cem\u003eC. albicans\u003c/em\u003e biofilms (strains SC5314, CA3153, and I49) in the presence of\u003cem\u003e \u003c/em\u003eSc3458 or with the industrial control (IC, CNCM I-3856) at 3 hpi. Data are normalized to the inhibitory effects of \u003cem\u003eS. cerevisiae \u003c/em\u003ealone and compared to neutrophils with \u003cem\u003eC. albicans\u003c/em\u003e alone. \u003cstrong\u003eB.\u003c/strong\u003e Neutrophil-mediated killing of \u003cem\u003eC. albicans\u003c/em\u003e (strains SC5314, CA3153, and I49) was assessed at 1 and 3 hpi with Sc3458 or with the industrial control (IC, CNCM I-3856). Data are normalized to \u003cem\u003eC. albicans\u003c/em\u003ewith neutrophils alone (100%). \u003cstrong\u003eC.\u003c/strong\u003eRepresentative images of mScarlet-tagged \u003cem\u003eC. albicans\u003c/em\u003e(strain CA3153) treated with neutrophils in the presence of Sc3458. Images show \u003cem\u003eC. albicans\u003c/em\u003e (red), neutrophil death/NETosis via Sytox Green staining (green), and merged phase contrast. Scale bars = 200 μm. \u003cstrong\u003eD.\u003c/strong\u003e Overgrowth of\u003cem\u003e C. albicans \u003c/em\u003estrains SC5314, CA3153, and I49 was monitored over 12 hours in the presence of neutrophils, with or without\u003cem\u003e S. cerevisiae \u003c/em\u003estrain 3458 or the industrial control (IC, CNCM I-3856), and quantified by Total Orange Object Integrated Intensity. \u003cstrong\u003eE. \u003c/strong\u003eNETosis\u003cstrong\u003e \u003c/strong\u003einduced by \u003cem\u003eC. albicans\u003c/em\u003e strains SC5314 (left), CA3153 (middle), and I49 (right) under the same conditions as panel D, measured over 6 hours. NETosis was quantified via Sytox Green fluorescence and levels were normalized to the total neutrophil count. Values represent mean ± standard deviation (s.d.) from n ≥ 7 donors (A), n ≥ 8 (B), n ≥ 6 (D), and n ≥ 9 (E, F), with dots representing individual donors. Statistical significance was determined using one-sample t-tests against a mean of 100 (A,B). Statistical significance was determined using a linear mixed effects model with a Holm-Šidák multiple comparisons test (D,E,). Significance: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/108a36ec13623963e3534160.png"},{"id":84772112,"identity":"b623e1a5-463f-4ef0-85d7-45dabd0c968c","added_by":"auto","created_at":"2025-06-17 08:16:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1588162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficacy of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e3458 in an \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e murine model of VVC. A.\u003c/strong\u003e Schematic representation of the experimental setup, including estradiol pre-treatment, vaginal \u003cem\u003eC. albicans\u003c/em\u003e infection, and subsequent intravaginal administration of Sc3458, the industrial control strain (IC, CNCM I-3856), or PBS. \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e bioluminescence imaging (BLI): Quantification of total photon flux in \u003cem\u003eC. albicans\u003c/em\u003e-infected mice treated with Sc3458, industrial control strain (IC, CNCM I-3856), or PBS, acquired on days 2, 4, 6, 8, and 10 post-infection. Photon flux values were calculated based on a region of interest (ROI) covering the entire vaginal area (n ≥ 7). \u003cstrong\u003eC.\u003c/strong\u003e Representative BLI images of mice from each treatment group corresponding to the time points in panel B. \u003cstrong\u003eD.\u003c/strong\u003e Cytokine and chemokine analysis of vaginal lavage fluids collected on day 10 from \u003cem\u003eC. albicans\u003c/em\u003e-infected mice treated with Sc3458, industrial control strain (IC, CNCM I-3856), or PBS. Data include pro-inflammatory IL-1 family cytokines (IL-1α, IL-1β), neutrophil-recruiting chemokines (CXCL1, CXCL5), T-cell associated cytokines (IL-17A, IL-22), and allergy-related markers (IL-4, CCL11). PBS-treated mice serve as uninfected baseline controls. \u003cstrong\u003eE.\u003c/strong\u003e Absolute numbers of neutrophils, macrophages, and eosinophils in vaginal lavage fluids from each treatment group on day 10 post-infection. PBS-treated mice served as uninfected baseline controls.\u003cstrong\u003e\u003cbr\u003e\nF. \u003c/strong\u003eHeatmap representation of normalized expression levels (median fluorescence intensity) of activation markers CD11b, CD11c, Ly6C, and Ly6G on eosinophils, compared to the PBS-only condition.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003cstrong\u003e:\u003c/strong\u003e Depending on data distribution, statistical comparisons were performed using either one-way ANOVA followed by Holm-Šidák multiple comparisons test (B, D, E) or Kruskal-Wallis test followed by Dunn’s multiple comparisons test (F). Comparisons were made against the \u003cem\u003eC. albicans\u003c/em\u003e-infected, PBS-treated control group (B, D, E) and against the uninfected, PBS-treated group (F). Significance: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. Schematic representation was created with BioRender.com.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/5c411ce13dc6427c56cd792b.png"},{"id":84773098,"identity":"a89d915e-af2e-498e-b26c-1a3aa036c32f","added_by":"auto","created_at":"2025-06-17 08:24:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1433002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic overview of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. cerevisiae-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emediated interference with pathogenicity and inflammation during VVC.\u003c/strong\u003e Illustration of the dual therapeutic potential of \u003cem\u003eS. cerevisiae\u003c/em\u003e (blue cells) in mitigating VVC. On the left, \u003cem\u003eC. albicans\u003c/em\u003e-driven (beige cells) VVC is characterized by a high fungal burden, activation of epithelial signaling pathways (PI3K/Akt, MAPK, NF-κB), and recruitment of dysfunctional, anergic neutrophils (neutrophils; green), leading to excessive inflammation and epithelial damage. On the right, \u003cem\u003eS. cerevisiae\u003c/em\u003e treatment reduces fungal load, prevents overactivation of epithelial signaling pathways, and attenuates inflammatory responses. Simultaneously, \u003cem\u003eS. cerevisiae\u003c/em\u003e enhances neutrophil antifungal activity, promoting efficient fungal clearance while minimizing tissue damage. This \u003cem\u003eS. cerevisiae\u003c/em\u003e-mediated modulation of host immunity and fungal virulence disrupts the pathological cycle of inflammation and symptoms associated with VVC. Figure was created with BioRender.com.\u003csup\u003e93-100\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/480ffccdc267758c489e09d6.png"},{"id":88449434,"identity":"e424a998-928c-4839-8619-ca87aba8cb8d","added_by":"auto","created_at":"2025-08-06 14:15:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21522520,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/f7e9ea59-5cb8-4e25-b5c2-ea8f65ed436c.pdf"},{"id":84772107,"identity":"bc872738-3f54-41ed-a1dc-7208f3dbdfae","added_by":"auto","created_at":"2025-06-17 08:16:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4586890,"visible":true,"origin":"","legend":"Supplementary materials","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6795973/v1/0963aac498d50a34efbb17c2.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003e\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e reduces vulvovaginal candidiasis severity through modulation of fungal pathogenicity and inflammatory responses\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVulvovaginal candidiasis (VVC), an infection of the vaginal mucosa caused by yeast of the genus \u003cem\u003eCandida\u003c/em\u003e, is the second most prevalent form of vaginitis, posing a significant health concern \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. VVC affects up to 75% of women at least once, regardless of their overall health status\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Moreover, approximately 7\u0026ndash;9% of women endure recurrent vulvovaginal candidiasis (RVVC), defined by three or more infections annually\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, contributing to an estimated global burden of 138\u0026nbsp;million cases per year\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. (R)VVC manifests distressing symptoms, such as white clumpy discharge, burning, redness, and itching in the vulva and vagina, and dyspareunia, significantly impacting mental well-being\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCandida albicans\u003c/em\u003e, the most prevalent causative species\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, induces VVC through hyperinflammation, driven by \u003cem\u003eCandida\u003c/em\u003e virulence and maintained by the innate immune system, resulting in compromised neutrophil-mediated fungal clearance\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. VVC manifests when the fungal burden exceeds an epithelial threshold, triggering epithelial pro-inflammatory signaling cascades that result in the release of inflammatory mediators\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. This process catalyzes downstream inflammatory responses that facilitate the recruitment of immune cells, primarily neutrophils\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the hyperinflammation and specific characteristics of the vaginal niche impair neutrophils' ability to effectively deploy their antifungal mechanisms, leading to tissue damage and perpetuating a cycle of inflammation and exacerbation of symptoms\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBeing a multifaceted disease, VVC presents multiple therapeutic opportunities, including antifungal drugs and vaccines still under research\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. While antifungals are central to RVVC management, prolonged use often fosters resistance\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Coupled with the complex nature of VVC, this leaves many patients insufficiently treated. Moreover, antifungals only indirectly impact inflammatory responses and fail to alleviate symptoms when non-self-limiting inflammation drives disease progression\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, alternative treatment strategies that tackle VVC\u0026rsquo;s multifactorial nature are urgently needed. Given the essential role of vaginal microbial communities in maintaining vaginal health, live microbial-biotherapeutics present a promising avenue\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Therefore, alternative treatment strategies that tackle VVC\u0026rsquo;s multifactorial nature are urgently needed. Given the essential role of vaginal microbial communities in maintaining vaginal health, live microbial-biotherapeutics (LBPs) present a promising avenue\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These are defined as live microorganisms intended for the prevention or treatment of disease\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and several \u003cem\u003eLactobacillaceae\u003c/em\u003e-based LBPs have shown efficacy in experimental and clinical VVC\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, as infections are highly patient-specific, treatment efficacy is not universal\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Although yeast are generally not dominant members of the vaginal microbiome, clinical research highlights reduced levels of \u003cem\u003eSaccharomyces\u003c/em\u003e spp. in women with RVVC compared to healthy individuals, making these yeasts intriguing candidates for further study\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eS. cerevisiae\u003c/em\u003e especially shows promise for use in patients undergoing antibiotic therapy, a common trigger for VVC development\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Recognized for its benefits in gastrointestinal disorders\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, one \u003cem\u003eS. cerevisiae\u003c/em\u003e strain has also shown efficacy in the context of VVC, demonstrating the ability to coaggregate with \u003cem\u003eC. albicans\u003c/em\u003e, inhibit its adherence to epithelial cells, suppress key virulence factors, and protect epithelial cells from \u003cem\u003eCandida\u003c/em\u003e-induced damage\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Its effects likely arise from mechanisms such as pathogen binding, production of antimicrobial peptides, trophic effects, and immune-modulatory properties\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we focused on the potential of \u003cem\u003eS. cerevisiae\u003c/em\u003e in counteracting \u003cem\u003eC. albicans\u003c/em\u003e-induced VVC in lab-based and murine models. We conducted a first-in-disease comprehensive screening of a broad library of \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates to assess their potential as therapeutic microbes against VVC. We explored the mechanisms through which \u003cem\u003eS. cerevisiae\u003c/em\u003e antagonizes \u003cem\u003eC. albicans\u003c/em\u003e pathogenicity and modulates immune responses, focusing on insights gained from multispecies transcriptomics combined with \u003cem\u003ein vitro\u003c/em\u003e infection models. To validate these findings in a physiologically relevant context, we treated mice with \u003cem\u003eS. cerevisiae\u003c/em\u003e in the context of a well-established VVC model. Through examining the tripartite interactions between \u003cem\u003eC. albicans\u003c/em\u003e, vaginal epithelial cells, and neutrophils\u0026mdash;both in \u003cem\u003ein vitro\u003c/em\u003e and in murine models\u0026mdash;we uncovered novel mechanistic insights into how \u003cem\u003eS. cerevisiae\u003c/em\u003e acts as a live biotherapeutic for VVC. \u003cem\u003eC. albicans\u003c/em\u003e virulence inhibition by physical constraint, metabolic interference and transcriptional reprogramming resulted in a dampened pro-inflammatory milieu and enhanced neutrophil-mediated fungal clearance, two critical determinants in the pathogenesis of symptomatic VVC. These findings establish a robust preclinical foundation for the translational development of \u003cem\u003eS. cerevisiae\u003c/em\u003e-based interventions in human VVC.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e \u003cb\u003estrain-specific inhibition of\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eGiven large strain-to-strain variability within microbial species\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, a screening was performed to identify \u003cem\u003eS. cerevisiae\u003c/em\u003e strains with inhibitory properties against \u003cem\u003eC. albicans\u003c/em\u003e, a key driver of VVC pathology\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We screened 70 diverse \u003cem\u003eS. cerevisiae\u003c/em\u003e strains, including food, lab strains, probiotics, beverages, and human sources (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eUsing assays for \u003cem\u003eC. albicans\u003c/em\u003e CA3153 growth inhibition in vagina-simulative medium (VSM) and RPMI, adhesion to A-431 vaginal epithelial cells (VECs), and filamentation inhibition, several effective \u003cem\u003eS. cerevisiae\u003c/em\u003e strains were identified (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Notably, inhibitory capacity was not linked to the strain's origin. From the 70 strains initially screened, the most promising candidates were shortlisted based on top performance in inhibiting \u003cem\u003eC. albicans\u003c/em\u003e virulence, with those showing consistent efficacy across multiple assays prioritized. Based on these criteria, strains 4609, 6883, 6886, 6888, 6894, 3458, 3471, 3076, and Y.00240 were selected (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Two additional control strains were also included: the lab strain S288c and the probiotic standard CNCM I-3856, currently marketed for gastrointestinal (\u003cem\u003eibSium\u0026reg;\u003c/em\u003e) and vaginal (\u003cem\u003eQuatreflora\u0026reg;\u003c/em\u003e) health\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e \u003cb\u003ereduces\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003epathogenicity on multiple levels.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the inhibitory potential of \u003cem\u003eS. cerevisiae\u003c/em\u003e against \u003cem\u003eC. albicans\u003c/em\u003e virulence in depth, we built on our extensive screening and focused on early infection processes across three \u003cem\u003eC. albicans\u003c/em\u003e isolates SC5314, CA3153, and I49. Growth of \u003cem\u003eC. albicans\u003c/em\u003e isolates in both VSM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and RPMI (\u003cb\u003eFig. S2A\u003c/b\u003e) was broadly inhibited by the selected \u003cem\u003eS. cerevisiae\u003c/em\u003e strains, reducing \u003cem\u003eC. albicans\u003c/em\u003e growth by up to 25\u0026ndash;50% within 6 hours and reaching up to 75% inhibition by 24 hours. Notably, specific strains - C(6886), F(3458), G(3471), and IC(CNCM I-3856) - achieved higher levels of inhibition at earlier time points. Moreover, the growth inhibition was robust across all tested \u003cem\u003eC. albicans\u003c/em\u003e isolates.\u003c/p\u003e \u003cp\u003eThe adhesion of \u003cem\u003eC. albicans\u003c/em\u003e to VECs is crucial for the initiation of infection, invading the epithelial cells and causing damage\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Simultaneous introduction of \u003cem\u003eS. cerevisiae\u003c/em\u003e reduced \u003cem\u003eC. albicans\u003c/em\u003e adhesion to VECs by ~\u0026thinsp;30%, while some strains displaced up to 50% of pre-adhered \u003cem\u003eC. albicans\u003c/em\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), disrupting established \u003cem\u003eC. albicans\u003c/em\u003e\u0026ndash;VEC interactions. Despite variability between \u003cem\u003eS. cerevisiae\u003c/em\u003e strains and individual repetitions, certain strains consistently inhibited adherence of all three \u003cem\u003eC. albicans\u003c/em\u003e isolates.\u003c/p\u003e \u003cp\u003eThe morphological transition to filamentous growth strengthens adhesion of \u003cem\u003eC. albicans\u003c/em\u003e and facilitates invasion and damage of epithelial cells\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This further prompts \u003cem\u003eC. albicans\u003c/em\u003e recognition and initiation of epithelial inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Specific \u003cem\u003eS. cerevisiae\u003c/em\u003e strains (A(4609), C(6886), F(3458), G(3471), and H(3076)) were able to inhibit (30\u0026ndash;50%) or delay hyphal growth in VSM after 3 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), though the effects were less pronounced in RPMI which by itself is a stronger stimulus for filamentation (\u003cb\u003eFig. S2B\u003c/b\u003e). While all \u003cem\u003eS. cerevisiae\u003c/em\u003e strains reduced \u003cem\u003eC. albicans\u003c/em\u003e growth in VSM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B), not all inhibited the morphological transition, and some, B(6883), I(Y.00240), and LC(S288c), even promoted hyphal growth.\u003c/p\u003e \u003cp\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e \u003cb\u003emitigates\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e\u003cb\u003e-induced epithelial damage and inflammation.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe inhibitory effect of \u003cem\u003eS. cerevisiae\u003c/em\u003e on \u003cem\u003eC. albicans\u003c/em\u003e growth, filamentation and adhesion to VECs could imply decreased epithelial tissue damage and inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eS. cerevisiae\u003c/em\u003e co-cultured reduced epithelial damage inflicted by \u003cem\u003eC. albicans\u003c/em\u003e, quantified by the release of the host cytoplasmic enzyme LDH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). While certain \u003cem\u003eS. cerevisiae\u003c/em\u003e strains - D(6888), E(6894), F(3458), G(3471) - markedly reduced cell damage, some - I(Y.00240), LC(S288c), and IC(CNCM I-3856) - had minimal effect or even exacerbated toxicity.\u003c/p\u003e \u003cp\u003eThrough the release of alarmins such as IL-1α, epithelial tissue damage can catalyze a pro-inflammatory state, activating neutrophils\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eC. albicans\u003c/em\u003e infection alone triggered minimal IL-1α release (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C), which was not significantly altered by protective \u003cem\u003eS. cerevisiae\u003c/em\u003e strains, suggesting that additional factors may regulate its production. MAPK pathways, activated by excessive fungal burden and hyphae, exacerbate VVC pathology by recruiting and activating neutrophils\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eS. cerevisiae\u003c/em\u003e strains that dampened fungal growth and filamentation also reduced IL-8 and GM-CSF responses in VECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, E), cytokines that enhance neutrophil activation, survival, and effector function (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Importantly, the probiotic \u003cem\u003eS. cerevisiae\u003c/em\u003e strain IC (CNCM I-3856) enhanced pro-inflammatory cytokine responses, amplifying signaling beyond the response to \u003cem\u003eC. albicans\u003c/em\u003e. Unlike other \u003cem\u003eS. cerevisiae\u003c/em\u003e strains, it also independently triggered pro-inflammatory responses in VECs in the absence of \u003cem\u003eC. albicans\u003c/em\u003e (\u003cb\u003eFig. S3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCluster analysis reveals superior\u003c/b\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e \u003cb\u003estrains for VVC treatment.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur data demonstrate that \u003cem\u003eS. cerevisiae\u003c/em\u003e strains can variably attenuate \u003cem\u003eC. albicans\u003c/em\u003e virulence and alleviate the epithelial immune responses. We aimed to identify a strain that consistently exhibited superior performance across all assays. Multivariate cluster analysis using the collective data from previous assays revealed two distinct clusters (k-means, k\u0026thinsp;=\u0026thinsp;2), explaining over 70% of the variance observed in the dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). This significant level of explained variance indicates a strong association between the identified clusters and the observed performance outcomes.\u003c/p\u003e \u003cp\u003eThe first cluster, comprised of the \u003cem\u003eS. cerevisiae\u003c/em\u003e strains that demonstrated superior efficacy in reducing \u003cem\u003eC. albicans\u003c/em\u003e virulence and modulating immune responses, included A(4609), B(6883), C(6886), E(6894), F(3458), G(3471), and H(3076), suggesting a robust live biotherapeutic potential. The second cluster comprised of strains that exhibited overall low performance: D(6888), I(Y.00240), LC(S288c), and IC(CNCM I-3856). Based on the consistently high performance across all readouts, \u003cem\u003eS. cerevisiae\u003c/em\u003e strain (F) 3458 (Sc3458) was selected for further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e \u003cb\u003edrives transcriptional reprogramming in\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003eand VECs.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMultispecies transcriptomics was performed to identify underlying mechanisms by which Sc3458 attenuates \u003cem\u003eC. albicans\u003c/em\u003e virulence and modulates epithelial inflammatory responses. Principal component analysis (PCA) revealed distinct \u003cem\u003eC. albicans\u003c/em\u003e gene expression at 24-hours post-infection (hpi) with slight overlap at 6 hpi, depending on the presence of Sc3458 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, VECs showed distinct clusters in the presence of Sc3458 at 24 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Despite the proximity of PCA clusters, particularly for \u003cem\u003eC. albicans\u003c/em\u003e at 24 hpi, significantly differentially expressed genes (DEGs) were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cb\u003eupper panel\u003c/b\u003e). PCA highlights major variability in gene expression between conditions, but overlapping clusters can still harbor substantial transcriptional differences. At 6 hpi, \u003cem\u003eC. albicans\u003c/em\u003e showed 9 DEGs (\u003cb\u003eFig. S4\u003c/b\u003e), but this increased to 211 DEGs at 24 hpi in response to Sc3458. For VECs, the impact of \u003cem\u003eS. cerevisiae\u003c/em\u003e was more pronounced at 24 hpi, with 1848 DEGs observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cb\u003elower panel\u003c/b\u003e). Together, the DEG counts and PCA results underscore that Sc3458 drives significant reprogramming of both \u003cem\u003eC. albicans\u003c/em\u003e and VEC gene expression, particularly at 24 hpi.\u003c/p\u003e \u003cp\u003eMore specifically for \u003cem\u003eC. albicans\u003c/em\u003e, a total of 131 genes were downregulated, while 80 genes were upregulated when \u003cem\u003eS. cerevisiae\u003c/em\u003e was present during infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Gene Ontology (GO) enrichment analysis revealed upregulation of genes related to \u0026ldquo;amino acid transport and metabolism\u0026rdquo; (\u003cem\u003eAGP2, ALP1, SER2\u003c/em\u003e) and \u0026ldquo;sulfur compound biosynthesis\u0026rdquo; (\u003cem\u003eSAM2, MET16, MET15, CYS3\u003c/em\u003e), reflecting metabolic shifts to counter nutrient competition and oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Increased expression of ion transport genes (\u003cem\u003eFRE10, CFL2, CFL5\u003c/em\u003e) suggests efforts to maintain ionic and redox balance under oxidative stress. Upregulated biosynthetic (\u003cem\u003eACS2, ACC1\u003c/em\u003e) and ribosomal genes (\u003cem\u003eGAR1, RPL39\u003c/em\u003e) indicate heightened protein synthesis demands to manage stress and support growth. These stress-induced adaptations highlight \u003cem\u003eC. albicans'\u003c/em\u003e attempt to reprogram its metabolism and homeostatic mechanisms to survive the antagonistic effects imposed by Sc3458 during infection.\u003c/p\u003e \u003cp\u003eThe analysis of downregulated genes revealed links to processes essential for \u003cem\u003eC. albicans\u003c/em\u003e biofilm development and cell-cell interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). GO term enrichment identified significant downregulation in genes involved in \u0026ldquo;single-species biofilm development\u0026rdquo; (both submerged and on inanimate substrates), \u0026ldquo;cell aggregation,\u0026rdquo; and \u0026ldquo;intraspecies interactions.\u0026rdquo; Key metabolic pathways for nutrient acquisition within the biofilm microniche were impacted, with genes involved in monosaccharide, hexose, and carbohydrate transport (\u003cem\u003eHGT13, GAL1, MAL31\u003c/em\u003e) downregulated, suggesting compromised biofilm integrity and cellular function. Genes related to adhesion (\u003cem\u003eTRY5, SUC1, ZCF28, SAP2/4/8, DSE1\u003c/em\u003e), morphogenesis (\u003cem\u003eHGC1, YAK1, CSA2, MAL2\u003c/em\u003e), and biofilm maturation (\u003cem\u003eSAP8, PGA6\u003c/em\u003e) were also suppressed, proposing significant disruption of biofilm structure and functionality.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eDirect interaction and metabolic activity of\u003c/strong\u003e \u003cstrong\u003eS. cerevisiae\u003c/strong\u003e \u003cstrong\u003eare essential for anti-biofilm effects on\u003c/strong\u003e \u003cstrong\u003eC. albicans.\u003c/strong\u003e \u003cem\u003eC. albicans\u003c/em\u003e biofilm formation on epithelial cells during VVC exceeds the epithelial threshold, triggering inflammatory responses and hindering neutrophil-mediated fungal clearance\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. While metabolic changes in \u003cem\u003eC. albicans\u003c/em\u003e induced by probiotics are known to reduce pathogenicity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, their specific effects on biofilm formation remain unclear. As biofilms play a key role in VVC pathogenesis, we investigated how Sc3458 impacts \u003cem\u003eC. albicans\u003c/em\u003e biofilm formation.\u003c/p\u003e\n\u003cp\u003eCorresponding with reduced \u003cem\u003eC. albicans\u003c/em\u003e growth and filamentation on VECs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), a broad downregulation of \u003cem\u003eC. albicans\u003c/em\u003e biofilm-related genes in response to the presence of Sc3458 is observed (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Accordingly, Z-stack microscopy proved that Sc3458 significantly reduced \u003cem\u003eC. albicans\u003c/em\u003e biofilm biomass on VECs during both early (24 hpi) and mature biofilm (48 hpi) stages (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Additionally, biofilm thickness was diminished by Sc3458 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, D). These inhibitory effects were comparable to those observed with the industrial control CNCM I-3856. We hypothesized that \u003cem\u003eS. cerevisiae\u003c/em\u003e might redistribute \u003cem\u003eC. albicans\u003c/em\u003e cells between the biofilm associated with VECs and cells in the supernatant, given the known aggregation tendencies of both species\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Interestingly, redistribution of cells from the biofilm to the supernatant was observed upon Sc3458 treatment (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). A similar redistribution pattern was observed for IC (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE), suggesting a conserved interference mechanism. While Sc3458 alone primarily remained in the supernatant, indicating weak adhesion to VECs (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF), the presence of \u003cem\u003eC. albicans\u003c/em\u003e resulted in its incorporation into the biofilm. Strikingly, Sc3458 treatment displaced \u003cem\u003eC. albicans\u003c/em\u003e into the supernatant while embedding itself within the \u003cem\u003eC. albicans\u003c/em\u003e biofilm, resulting in fewer \u003cem\u003eC. albicans\u003c/em\u003e cells being directly associated with the VECs and capable of inflicting damage and eliciting inflammatory responses.\u003c/p\u003e\n\u003cp\u003eGiven the gene expression patterns in \u003cem\u003eC. albicans\u003c/em\u003e indicating cell aggregation and metabolic stress, we investigated whether the inhibitory effects of Sc3458 depend on direct contact and metabolically active cells. Using transwells to separate Sc3458 from \u003cem\u003eC. albicans\u003c/em\u003e, allowing nutrient and molecule exchange, completely abolished the inhibition of biofilm formation (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). Similarly, heat-inactivated Sc3458 cells failed to inhibit biofilm formation (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). These findings indicate that biofilm inhibition by Sc3458 depends on both direct contact and cellular metabolism. The IC strain showed similar contact-dependent behavior (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG), reinforcing the robustness of this mechanism (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e \u003cstrong\u003esuppresses pro-inflammatory responses and neutrophil activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess how Sc3458 treatment modulates inflammatory responses, we conducted a detailed analysis of the epithelial transcriptional changes, revealing significant reprogramming at 24 hpi (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). A total of 1581 genes were downregulated and 267 upregulated (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). GO enrichment analysis identified enhanced biological processes, including mitochondrial function, DNA repair, and RNA transport and processing, in response to Sc3458 treatment (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Although direct and well-established links to VVC pathology remain elusive, the GO-terms suggest a general activation of integral cellular maintenance and adaptive response pathways.\u003c/p\u003e\n\u003cp\u003eThe GO enrichment analysis highlighted many downregulated processes (\u003cstrong\u003eFig. S5\u003c/strong\u003e). We focused particularly on those relevant in the context of VVC, such as responses to stimuli, cell death, and inflammatory responses (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). Key affected pathways included \u0026ldquo;cell surface receptor signaling\u0026rdquo;, \u0026ldquo;environmental and abiotic stimulus responses\u0026rdquo;, and \u0026ldquo;regulation of apoptotic and inflammatory signaling\u0026rdquo;. Notably, critical immune pathways \u0026mdash;Nuclear Factor kappa B (NF-\u0026kappa;B), Mitogen-Activated Protein Kinase (MAPK), and Phosphoinositide 3-Kinase/Protein Kinase B (PI3K/Akt) cascades\u0026mdash; were downregulated, alongside Wnt signaling, phagocytosis, and autophagy regulation. Given the importance of MAPK, NF-\u0026kappa;B, and PI3K/Akt pathways in epithelial responses to \u003cem\u003eC. albicans\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, we further analyzed specific DEGs within these pathways. Key genes in the MAPK pathway (\u003cem\u003eMKK7\u003c/em\u003e and \u003cem\u003eMLK3\u003c/em\u003e) were downregulated, critical for activating JNK and p38 (\u003cstrong\u003eFig. S6\u003c/strong\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eMK2\u003c/em\u003e and \u003cem\u003eICAM1\u003c/em\u003e, involved in inflammatory signaling, were also downregulated. MK2 regulates cytokine production \u003cem\u003evia\u003c/em\u003e the p38 MAPK pathway, while ICAM1 facilitates immune cell adhesion and recruitment\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eERK1\u003c/em\u003e, part of a separate MAPK branch, was also downregulated, a pathway associated with processes such as cell proliferation and stress responses to \u003cem\u003eC. albicans\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In the NF-\u0026kappa;B pathway, \u003cem\u003ep65\u003c/em\u003e, \u003cem\u003eRelB\u003c/em\u003e, and \u003cem\u003eNEMO\u003c/em\u003e were all downregulated, components essential for cytokine transcription and anti-apoptotic signaling\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Similarly, \u003cem\u003eAKT1\u003c/em\u003e, \u003cem\u003eAKT2\u003c/em\u003e, and \u003cem\u003eIRS1\u003c/em\u003e, part of the PI3K/Akt pathway, were downregulated, affecting survival signaling\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Reduced expression of \u003cem\u003eTLR2\u003c/em\u003e and \u003cem\u003eFGFR1\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e indicated diminished immune activation and growth factor signaling. Downregulation of \u003cem\u003eeNOS\u003c/em\u003e, \u003cem\u003eFOXO1\u003c/em\u003e, and \u003cem\u003eFOXO4\u003c/em\u003e further supports a shift in apoptosis regulation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTranscriptional profiling revealed upregulation of \u003cem\u003eIL1B\u003c/em\u003e in \u003cem\u003eC. albicans\u003c/em\u003e-infected VECs exposed to \u003cem\u003eS. cerevisiae\u003c/em\u003e (\u003cstrong\u003eFig. S6\u003c/strong\u003e). Excessive IL-1\u0026beta; production hyperactivates neutrophils, causing tissue damage without effectively clearing the pathogen, a hallmark of severe VVC. Unlike macrophages, VECs produce limited inflammasome-dependent IL-1\u0026beta;, and no IL-1\u0026beta; responses have been detected upon \u003cem\u003eC. albicans\u003c/em\u003e infection of VECs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. By assessing IL-1\u0026beta; release by human monocyte-derived macrophages (hMDMs)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, we found that Sc3458 rather reduced IL-1\u0026beta; responses (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). In contrast, the IC strain increased IL-1\u0026beta; secretion (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD), indicating that it reinforces inflammation across both epithelial (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) and macrophage compartments. These data highlight a key functional distinction: while IC broadly amplifies inflammation, Sc3458 appears to reduce inflammation across epithelial cells and macrophages.\u003c/p\u003e\n\u003cp\u003eNext, we validated how the downregulation of pro-inflammatory MAPK and NF-\u0026kappa;B pathways corresponded to reduced downstream neutrophil activation by the epithelial cells. Supernatants from epithelial infections with Sc3458 led to slightly lower IL-8 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE,F\u003cstrong\u003e)\u003c/strong\u003e secretion and reduced ROS release by neutrophils (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). Both effects were also seen with the IC strain, albeit with strain-specific variability. Although not all changes were statistically significant, an overall trend of reduced neutrophil activation was evident. In line with this, neutrophils exposed to supernatants from \u003cem\u003eC. albicans\u003c/em\u003e-infected VECs treated with Sc3458 exhibited altered surface marker expression (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH). CD11b, an integrin involved in fungal recognition and recruitment, was downregulated, alongside CD66b, a marker of secondary granule mobilization. CD35 also decreased, suggesting reduced degranulation. In contrast, CD16 expression was elevated, consistent with enhanced neutrophil viability or antimicrobial readiness. Notably, CD62L, typically shed during activation, was preserved, indicating a less activated phenotype. Moderate reduction in CXCR2, which is internalized upon chemokine engagement, pointed to recent activation and potential migratory readiness. These expression patterns were broadly comparable to those induced by the IC strain, suggesting that, despite differences in upstream modulation of inflammation, both strains elicit a similarly tempered neutrophil phenotype. Nevertheless, responses varied depending on the \u003cem\u003eC. albicans\u003c/em\u003e isolate used, with CA3153 and I49 generally inducing weaker pro-inflammatory signaling. Importantly, Sc3458 and IC did not affect neutrophil chemotaxis across the endothelial\u0026ndash;epithelial barrier in response to \u003cem\u003eC. albicans\u003c/em\u003e-infected VECs (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e\n\u003cp\u003eCollectively, these findings indicate that Sc3458 modulates neutrophil activation by dampening upstream epithelial signaling, while preserving neutrophil recruitment to the site of infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e \u003cstrong\u003epromotes neutrophil-mediated\u003c/strong\u003e \u003cstrong\u003eC. albicans\u003c/strong\u003e \u003cstrong\u003eclearance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that Sc3458 reduced neutrophil activation downstream of \u003cem\u003eC. albicans\u003c/em\u003e-infected VECs, we next assessed its direct effects on neutrophil-mediated fungal clearance, known to be impaired during VVC\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to inhibiting \u003cem\u003eC. albicans\u003c/em\u003e biofilm formation (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), Sc3458 treatment enhanced neutrophil-mediated clearance of \u003cem\u003eC. albicans\u003c/em\u003e biofilm-associated cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). This led to an additive effect, with \u003cem\u003eC. albicans\u003c/em\u003e biofilm biomass reduced by 30\u0026ndash;50% beyond the inhibition observed with Sc3458 treatment alone. To further evaluate this interaction, we assessed neutrophil-mediated killing at early time points. Sc3458 significantly enhanced fungal killing at both 1 and 3 hpi, reducing fungal viability up to ~\u0026thinsp;40% compared to neutrophils alone (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). A similar enhancement of neutrophil-mediated clearance was observed with strain IC, suggesting that this trait can be shared across \u003cem\u003eS. cerevisiae\u003c/em\u003e strains. The enhanced killing was consistent for \u003cem\u003eC. albicans\u003c/em\u003e isolates SC5314 and CA3153. However, for isolate I49, which previously showed reduced virulence and delayed immune activation, the neutrophil-mediated killing effect was absent at 1 hpi.\u003c/p\u003e\n\u003cp\u003eUsing live-cell analysis, we simultaneously assessed fungal overgrowth, neutrophil death and NETosis (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Quantification of mScarlet-tagged \u003cem\u003eC. albicans\u003c/em\u003e revealed significant reductions in \u003cem\u003eC. albicans\u003c/em\u003e burden upon Sc3458 and IC treatment overtime, starting 2 hpi (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). The visualization of treatment after 6 hours helped us to understand the interactions between neutrophils and \u003cem\u003eC. albicans\u003c/em\u003e, in the presence of \u003cem\u003eS. cerevisiae\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). Images show enhanced neutrophil clustering and reduced \u003cem\u003eC. albicans\u003c/em\u003e overgrowth upon Sc3458 treatment compared to neutrophils cultured with \u003cem\u003eC. albicans\u003c/em\u003e alone, where fungal persistence was more evident. Interestingly, improved \u003cem\u003eC. albicans\u003c/em\u003e killing and reduced overgrowth were not accompanied by significant changes in overall neutrophil cell death (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eS). Similarly, NETosis events were not enhanced by the presence of Sc3458 or strain IC (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE), suggesting that enhanced fungal clearance occurs through mechanisms independent of NETosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eS. cerevisiae\u003c/strong\u003e \u003cstrong\u003e3458 reduces\u003c/strong\u003e \u003cstrong\u003eC. albicans\u003c/strong\u003e \u003cstrong\u003eburden and attenuates local inflammation in a murine model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the pathogenicity attenuating and immunomodulatory effects observed \u003cem\u003ein vitro\u003c/em\u003e translated to an improved disease outcome, we treated mice with Sc3458 in the context of a well-established murine VVC model. Mice in a pseudoestrous state were intravaginally administered Sc3458, the industrial control strain (IC), or PBS (10 \u0026micro;L of 10⁸ cells/mL per mouse) one day after infection with \u003cem\u003eC. albicans\u003c/em\u003e (10 \u0026micro;L of 10⁷ cells/mL per mouse) (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). Longitudinal bioluminescence imaging (BLI) of the bioluminescent \u003cem\u003eC. albicans\u003c/em\u003e reporter revealed a significant reduction in fungal burden following treatment with Sc3458. From day 6 onward, mice treated with Sc3458 exhibited a sustained decrease in \u003cem\u003eC. albicans\u003c/em\u003e vaginal burden, while PBS-treated controls increased in fungal burden (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, C; \u003cstrong\u003eFig. S8\u003c/strong\u003e). The IC strain showed a slight early reduction in fungal burden compared to \u003cem\u003eC. albicans\u003c/em\u003e alone; however, this trend was not statistically significant and was not sustained beyond day 6, highlighting a lack of durable antifungal activity compared to Sc3458 (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). Along with the high fungal burden in PBS-treated controls 10-days post infection we observed an IL-1-(IL-1\u0026alpha; and IL-1\u0026beta;) dominated inflammatory response in vaginal lavages that has been extensively associated with the dysregulated inflammatory state driving VVC severity (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). Sc3458 treated mice, however, did not exhibit this characteristic pro-inflammatory response. Given the 10-day infection, adaptive immune responses are also activated. Even though controversial regarding their contribution to VVC\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, treatment with Sc3458 dampened both Th17- and Th22-associated cytokines IL-17A and IL-22 (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). Furthermore, type 2 immunity- and allergy-related cytokines, including IL-4, IL-9, IL-31, and the eosinophil-recruiting chemokine CCL11 (eotaxin-1), were significantly reduced in the Sc3458-treated group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cstrong\u003eFig. S8\u003c/strong\u003e), suggesting a potential decrease in mucosal irritation and symptom severity.\u003c/p\u003e\n\u003cp\u003eInterestingly, while most inflammatory mediators were diminished in the vaginal niche following Sc3458 treatment, neutrophil-chemokines such as CXCL2 remained elevated and CXCL1, and CXCL5 were even significantly higher (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cstrong\u003eFig. S9\u003c/strong\u003e), indicating a preserved or possibly enhanced chemotactic signal for neutrophil recruitment. Whilst the industrial control also decreased levels of several inflammatory mediators, similar to our observation \u003cem\u003ein vitro\u003c/em\u003e, it increased IL-1\u0026beta; release and showed an even stronger type 2 phenotype reflected in IL-4 and CCL11 levels. These observations reaffirm the unique immunomodulatory potential of Sc3458 compared to the IC strain (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cstrong\u003eFig. S9\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFlow cytometry of vaginal lavage samples revealed minimal differences in total neutrophil and macrophage counts across infected groups (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE), indicating that immune cell recruitment was not substantially altered by treatment. However, only Sc3458-treated mice exhibited reduced fungal burden, suggesting enhanced neutrophil functionality despite similar cell numbers. Eosinophil counts remained stable, yet Sc3458 downregulated CD11b and CD11c expression on eosinophils (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF). While these integrins are often used as activation markers, they also play direct roles in pathogen recognition and cytokine signaling in eosinophils\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The observed downregulation is consistent with reduced type 2 inflammatory cytokines and may reflect a shift toward a less activated, less tissue-damaging phenotype.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study establishes the therapeutic potential of a specific \u003cem\u003eS. cerevisiae\u003c/em\u003e strain in the context of \u003cem\u003eC. albicans\u003c/em\u003e-driven VVC. Through systematic screening, we identified \u003cem\u003eS. cerevisiae\u003c/em\u003e 3458, originally isolated from the vaginal mycobiota, as highly capable of inhibiting \u003cem\u003eC. albicans\u003c/em\u003e growth, adhesion, and hyphal morphogenesis. This strain further set itself apart in the ability to reduce epithelial damage and inflammatory responses. Transcriptomic profiling and \u003cem\u003ein vitro\u003c/em\u003e infection models revealed that Sc3458 attenuates \u003cem\u003eC. albicans\u003c/em\u003e biofilm formation and suppresses pro-inflammatory epithelial signaling (MAPK, NF-κB). Additionally, it enhanced neutrophil-mediated fungal clearance \u003cem\u003ein vitro\u003c/em\u003e. In a murine model of VVC, treatment with Sc3458 significantly reduced fungal burden while steering from IL-1-driven hyperinflammation and type-2 allergic responses. Collectively, this underscores Sc3458\u0026rsquo;s translational relevance as a live biotherapeutic candidate.\u003c/p\u003e \u003cp\u003eEffective and affordable treatments for VVC are urgently needed due to its high prevalence and the increasing incidence of treatment failures driven by rising antifungal resistance\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Nevertheless, a universal cure is challenging due to heterogeneous predisposing factors, including genetic predisposition affecting immune responses, \u003cem\u003eC. albicans\u003c/em\u003e virulence, and personal habits\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Despite this complexity, the prevailing view is that while hyperactivation of the epithelial cells and neutrophils play a major role in the onset of VVC, \u003cem\u003eC. albicans\u003c/em\u003e virulence is equally crucial in driving disease progression\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To address these multifactorial drivers, we explored \u003cem\u003eS. cerevisiae\u003c/em\u003e as a live biotherapeutic targeting multiple VVC-related factors\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. We maintained consistent \u003cem\u003eS. cerevisiae\u003c/em\u003e-to-\u003cem\u003eC. albicans\u003c/em\u003e ratios, creating conditions that reflect a balanced interaction rather than overwhelming dominance, allowing us to evaluate its therapeutic efficacy under more equal conditions.\u003c/p\u003e \u003cp\u003e \u003cem\u003eS. cerevisiae\u003c/em\u003e has proven itself as a promising probiotic across diverse niches, combating pathogens and modulating immune responses\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, our screening revealed that many previously characterized probiotic isolates were ineffective as live biotherapeutics against \u003cem\u003eC. albicans\u003c/em\u003e in the VVC context. No clear correlation between niche specificity and antifungal activity emerged, likely due to strain-specific differences shaped by habitat-related idiosyncrasies, such as the microbial environment, host factors, and nutrient availability\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. While the two most effective isolates, 3458 and 3471, originated from vaginal environments, other vaginal isolates showed limited efficacy. This underscores that habitat origin alone does not predict performance as a live biotherapeutic, reflecting the complexity of strain-specific adaptations.\u003c/p\u003e \u003cp\u003eSc3458 potently attenuated multiple \u003cem\u003eC. albicans\u003c/em\u003e virulence traits, including proliferation, adhesion to epithelial surfaces, and the yeast-to-hypha transition. Given the multifactorial nature of effects against \u003cem\u003eC. albicans\u003c/em\u003e and inflammation, it is unlikely that a single mechanism underlies this inhibition. Although early time points like 6 hpi could capture direct responses, only a small number of genes were differentially expressed, and these are not established virulence factors. Genes such as \u003cem\u003eCSA2\u003c/em\u003e, \u003cem\u003eADH2\u003c/em\u003e, and \u003cem\u003eFMN1\u003c/em\u003e are primarily involved in metabolic or housekeeping functions\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In contrast, the broader transcriptional changes at 24 hpi, while potentially shaped by secondary effects, offer more insight into the cellular response to \u003cem\u003eS. cerevisiae\u003c/em\u003e treatment and highlight relevant functional pathways. Metabolic interference, previously described for lactobacilli, is a plausible contributor\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Transcriptomic profiling revealed upregulation of genes associated with amino acid biosynthesis (\u003cem\u003eMET1, MET2, LYS4, TRP4\u003c/em\u003e), alternative carbon metabolism (\u003cem\u003eACS1, GPD2\u003c/em\u003e), and the TCA cycle (\u003cem\u003eACO2, ACC1\u003c/em\u003e), consistent with nutrient stress in \u003cem\u003eC. albicans\u003c/em\u003e. While \u003cem\u003eS. cerevisiae\u003c/em\u003e is also known to secrete antifungal metabolites such as phenylethanol and tryptophol\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, the inhibitory effects we observed were absent or markedly diminished when direct contact between organisms was prevented. Based on the results of both transcriptomic and contact-dependence assays, we conclude that inhibition is not solely driven by diffusible metabolic products. Rather, contact appears to facilitate localized nutrient depletion. This dependence on contact is a trait of \u003cem\u003eS. cerevisiae\u003c/em\u003e previously proven as \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e co-aggregate through \u003cem\u003eALS\u003c/em\u003e gene-mediated interactions\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, imposing physical constraints on \u003cem\u003eC. albicans\u003c/em\u003e and intensifying metabolic competition. Additionally, the loss of \u003cem\u003eS. cerevisiae\u0026rsquo;s\u003c/em\u003e inhibitory effects upon heat inactivation suggests that it depends on metabolically active cells. However, as heat inactivation also alters cell surface properties\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, the observed \u003cem\u003eC. albicans\u003c/em\u003e transcriptional reprogramming may result from both metabolic competition and altered cell-cell interactions, warranting further investigation.\u003c/p\u003e \u003cp\u003eBuilding on this, transcriptomic profiling revealed that \u003cem\u003eS. cerevisiae\u003c/em\u003e interferes with \u003cem\u003eC. albicans\u003c/em\u003e biofilm formation on epithelial cells through both physical disruption (downregulation of \u003cem\u003eTRY5\u003c/em\u003e, \u003cem\u003eSUC1\u003c/em\u003e, \u003cem\u003eSAP8, HGC1, YAK1\u003c/em\u003e) and metabolic interference (downregulation of \u003cem\u003eHGT13, GAL1, MAL31\u003c/em\u003e). We found that \u003cem\u003eS. cerevisiae\u003c/em\u003e integrated into \u003cem\u003eC. albicans\u003c/em\u003e biofilms on epithelial cells, reducing biofilm density and displacing \u003cem\u003eC. albicans\u003c/em\u003e cells toward the vaginal lumen, where clearance is likely enhanced\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. This resembles \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e\u0026ndash;mediated displacement of \u003cem\u003eC. albicans\u003c/em\u003e from intestinal epithelia\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The downregulation of adhesion, morphogenesis, and biofilm genes in \u003cem\u003eC. albicans\u003c/em\u003e suggests structural repression, likely driven by \u003cem\u003eS. cerevisiae\u003c/em\u003e-induced metabolic competition. This supports a dual mechanism where metabolic and physical interference jointly disrupt biofilm integrity. While this trait has been reported in other contexts, it is novel in VVC. By disrupting biofilm structural integrity, \u003cem\u003eS. cerevisiae\u003c/em\u003e diminishes the biofilm\u0026rsquo;s protective properties against treatment and immune cell infiltration\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. This weakening may enhance the efficacy of conventional antifungal therapies, providing a potential avenue for synergistic treatment strategies\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCollectively, the multifaceted inhibitory mechanisms of \u003cem\u003eS. cerevisiae\u003c/em\u003e directly against \u003cem\u003eC. albicans\u003c/em\u003e already position it as a promising therapeutic candidate for \u003cem\u003eC. albicans\u003c/em\u003e-induced VVC.\u003c/p\u003e \u003cp\u003eWhile \u003cem\u003eC. albicans\u003c/em\u003e virulence is a key factor in VVC pathology, a dysregulated inflammatory response drives symptom severity\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. This is supported by elevated activation of key inflammatory signaling pathways (NF-κB, c-Fos, and p38) in both symptomatic and asymptomatic women colonized with hyphae-forming \u003cem\u003eC. albicans\u003c/em\u003e strains\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. We observed that reductions in \u003cem\u003eC. albicans\u003c/em\u003e virulence by Sc3458 were accompanied by dampened epithelial immune responses. The reduced \u003cem\u003eC. albicans\u003c/em\u003e colonization and hyphal morphology were associated with reduced activation of MAPK and NF-κB pathways, both critical for driving pro-inflammatory cytokine production in VECs\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Notably, \u003cem\u003eS. cerevisiae\u003c/em\u003e has been shown to modulate these same pathways in other host environments\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, suggesting a conserved mechanism of immune modulation. Within the NF-κB pathway, reduced expression of key components such as \u003cem\u003eRELA\u003c/em\u003e (p65), \u003cem\u003eRELB\u003c/em\u003e, and \u003cem\u003eNEMO\u003c/em\u003e was observed, potentially impairing pro-inflammatory cytokine transcription \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Similarly, downregulation of MAPK components, including \u003cem\u003eMKK7\u003c/em\u003e and \u003cem\u003eMLK3\u003c/em\u003e, suggests diminished JNK and p38 pathway activation\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, while reduced \u003cem\u003eERK1\u003c/em\u003e expression, associated with cell proliferation and stress responses, indicates decreased MAPK signaling overall. This reduction in MAPK signaling could decrease pro-inflammatory cytokine production, potentially mitigating mucosal inflammation during VVC\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Furthermore, \u003cem\u003eS. cerevisiae\u003c/em\u003e presence downregulated PI3K/Akt pathway components, including \u003cem\u003eAKT1\u003c/em\u003e, \u003cem\u003eAKT2\u003c/em\u003e, and \u003cem\u003eIRS1\u003c/em\u003e, further supporting an overall dampening of epithelial inflammatory signaling. Downregulation of pattern recognition and growth factor receptors such as \u003cem\u003eTLR2\u003c/em\u003e and \u003cem\u003eFGFR1/4\u003c/em\u003e, may further contribute to limiting immune activation and epithelial stress responses\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. While transcriptional changes may not directly reflect protein activity, the transcriptional shifts to a less inflammatory state align well with reduced fungal burden, inhibition of the yeast-to-hypha transition, decreased biofilm formation and inflammatory cytokine release from VECs. This scenario likely lowers IL-1 axis cytokines that drive uncontrolled neutrophil activation in VVC\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Correspondingly, we observed reduced epithelial damage alongside decreased IL-1α release. Similarly, monocyte-derived macrophages also showed reduced IL-1β responses upon Sc3458 presence. Given that these responses are critically dependent on the \u003cem\u003eC. albicans\u003c/em\u003e toxin candidalysin\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e, it is plausible that \u003cem\u003eS. cerevisiae\u003c/em\u003e interferes with toxin delivery, thereby mitigating inflammation and cell death\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Importantly, \u003cem\u003ein vivo\u003c/em\u003e Sc3458 treatment similarly suppressed IL-1 responses, while neutrophil chemoattractants such as CXCL1 and CXCL5 remained present or elevated. Despite slightly reduced neutrophil numbers, fungal burden was significantly lowered, suggesting that Sc3458 enhances neutrophil efficacy while mitigating IL-1-driven hyperinflammation, a hallmark of dysfunctional immunity in VVC\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. VVC is suggested to involve type 2 immunity, with IL-4 implicated in the mucosal allergy-like response seen in severe cases\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Our data demonstrate that Sc3458 markedly reduced the levels of both IL-4 and CCL11 in vaginal lavage fluid, alongside a reduction in IL-31, a cytokine associated with pruritus. Together, these findings point to Sc3458's potential to also alleviate symptomatic infection by moderating allergic inflammation. On top of that, while innate immunity is the primary defense against \u003cem\u003eC. albicans\u003c/em\u003e in the vagina, the extended \u003cem\u003ein vivo\u003c/em\u003e timeline allowed us to assess adaptive responses. Sc3458 treatment led to a marked reduction in IL-17A and IL-22 levels, indicating suppression of Th17-mediated inflammation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Although IL-17 and IL-22 are not strictly required for fungal control in all VVC models, their overactivation can drive mucosal inflammation. Notably, controlled elevation of IL-22 has been linked to protection through antimicrobial peptide induction\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Thus, Sc3458 may fine-tune Th17 responses, limiting immunopathology while maintaining effective fungal clearance. In contrast, the industrial control strain exacerbated inflammation, inducing higher levels of IL-1α, IL-1β, and IL-4. This indicates that IC promotes both hyperinflammation and mucosal allergy responses, which could aggravate symptoms.\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eC. albicans\u003c/em\u003e load and epithelial signaling are reduced by \u003cem\u003eS. cerevisiae\u003c/em\u003e, treatment is unlikely to begin immediately at infection onset, allowing time for dysregulated non-self-limiting neutrophil-mediated inflammation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Consequently, \u003cem\u003eS. cerevisiae\u0026rsquo;\u003c/em\u003es effect on VVC pathology may remain limited without directly influencing neutrophil activation and efficacy. We observed reduced epithelial-mediated neutrophil activation in the presence of \u003cem\u003eS. cerevisiae in vitro\u003c/em\u003e, including lower IL-8 and ROS levels, which are associated with heightened inflammation and exacerbated VVC symptoms\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Neutrophil surface marker expression suggested an altered activation state. Downregulation of CD35 and CD66b suggested reduced degranulation and release of tissue-damaging enzymes in response to \u003cem\u003eC. albicans\u003c/em\u003e-infected VECs\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. In contrast, CD62L, typically shed during activation, was preserved, indicating a less activated neutrophil phenotype\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Conversely, the chemokine receptor CXCR2 which is internalized upon chemokine sensing, showed moderate downregulation, supporting recent or active neutrophil recruitment\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. While CD16, linked to antimicrobial functionality, was upregulated\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Together, these \u003cem\u003ein vitro\u003c/em\u003e findings highlight that \u003cem\u003eS. cerevisiae\u003c/em\u003e tempers alters the neutrophil activation state while preserving the ability to effectively counteract the infection. Importantly, CD11b\u0026mdash;an integrin involved in neutrophil adhesion, recruitment, and recognition of \u003cem\u003eC. albicans\u003c/em\u003e antigens such as Pra1 and β-glucan\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e\u0026mdash;was also decreased, pointing to diminished activation potential by direct fungal recognition. While \u003cem\u003eS. cerevisiae\u003c/em\u003e had no striking effects on macrophage and granulocyte infiltration \u003cem\u003ein vivo\u003c/em\u003e, the activation state of eosinophils, particularly their elevated integrin expression, was congruent with human neutrophils, decreased in the presence of \u003cem\u003eS. cerevisiae\u003c/em\u003e. Beyond being highly upregulated on eosinophils by fungal exposure\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, CD11b/c are both antifungal recognition receptors\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e, and eosinophils heavily rely on CD11b for release of allergy-promoting molecules when engaging fungi \u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e,\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. This indicates that \u003cem\u003eS. cerevisiae\u003c/em\u003e treatment can moderate granulocytic inflammation of both neutrophils and eosinophils.\u003c/p\u003e \u003cp\u003eDespite dampening granulocyte activation, surprisingly, the treatment with \u003cem\u003eS. cerevisiae\u003c/em\u003e significantly enhanced neutrophil killing of \u003cem\u003eC. albicans\u003c/em\u003e and suppressed its overgrowth, without inducing excessive NET formation or compromising neutrophil viability. That \u003cem\u003eS. cerevisiae\u003c/em\u003e facilitates better killing, while even dampening neutrophil ROS, argues for a more directed antifungal response and less collateral damage\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Enhanced \u003cem\u003eC. albicans\u003c/em\u003e clearance may stem from improved recognition of the fungus, enabling more effective neutrophil responses, such as precise NET formation or enhanced phagocytosis. While \u003cem\u003eC. albicans\u003c/em\u003e masks β-glucans with mannoproteins to evade detection, \u003cem\u003eS. cerevisiae\u003c/em\u003e exposes β-glucans, activating Dectin-1-mediated neutrophil responses and countering immune evasion, enhancing neutrophil phagocytosis, oxidative burst, and antifungal activity\u003csup\u003e\u003cspan additionalcitationids=\"CR86 CR87\" citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e. Although it remains unclear whether \u003cem\u003eS. cerevisiae\u003c/em\u003e itself is phagocytosed to a significant extent, it does not appear to act as a harmful distraction for immune cells. Instead, its exposed β-glucans may prime or potentiate antifungal responses, thereby contributing to enhanced \u003cem\u003eC. albicans\u003c/em\u003e clearance. Additionally, \u003cem\u003eC. albicans\u003c/em\u003e metabolic stress in the presence of \u003cem\u003eS. cerevisiae\u003c/em\u003e may increase vulnerability to oxidative stress through impairing its ability to detoxify ROS \u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e. Further, by inhibiting filamentation, \u003cem\u003eS. cerevisiae\u003c/em\u003e enables neutrophils to prioritize efficient antimicrobial mechanisms like phagocytosis and intracellular killing over tissue-damaging degranulation and oxidative burst\u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e,\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eS. cerevisiae\u003c/em\u003e treatment enhanced neutrophil-mediated \u003cem\u003eC. albicans\u003c/em\u003e biofilm clearance. While the underlying mechanism remains to be fully elucidated, it may involve disruption of biofilm-associated barriers\u0026mdash;including extracellular matrix density, hyphal architecture, and altered PAMP expression\u0026mdash;that typically impair immune recognition and contribute to fungal persistence\u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e. As previously highlighted, enhanced neutrophil efficacy was also evident \u003cem\u003ein vivo\u003c/em\u003e: despite slightly reduced neutrophil numbers and a dampened IL-1\u0026ndash;driven inflammatory environment, Sc3458 treatment led to a significant reduction in \u003cem\u003eC. albicans\u003c/em\u003e burden. This supports the notion that Sc3458 not only modulates neutrophil activation but also enhances their antifungal function in a physiologically relevant setting. Interestingly, while the industrial control strain also improved neutrophil-mediated \u003cem\u003eC. albicans\u003c/em\u003e killing \u003cem\u003ein vitro\u003c/em\u003e, our \u003cem\u003ein vivo\u003c/em\u003e findings revealed only transient antifungal effects, accompanied by a pronounced pro-inflammatory and allergic response. These contrasting outcomes underscore the strain-specific immunomodulatory properties of \u003cem\u003eS. cerevisiae\u003c/em\u003e. Additionally, given the physiological and microbiome differences between mice and humans, further studies, particularly clinical studies, are needed to validate these findings and elucidate the underlying pathways driving neutrophil modulation.\u003c/p\u003e \u003cp\u003eOur broad screening of \u003cem\u003eS. cerevisiae\u003c/em\u003e isolates sets Sc3458 apart from other strains as a uniquely effective candidate therapeutic agent. We highlight the potential of Sc3458 as a dual-action VVC treatment, uniquely targeting both \u003cem\u003eC. albicans\u003c/em\u003e pathogenicity and modulating immune responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). By reducing fungal load, \u003cem\u003eS. cerevisiae\u003c/em\u003e alleviates epithelial stimulation, specifically the pro-inflammatory IL-1 axis and prevents excessive neutrophil activation, thereby mitigating inflammation and epithelial damage. Simultaneously, it recalibrates neutrophil antifungal mechanisms, enhancing fungal clearance without reliance on tissue-damaging responses such as excessive ROS production. Collectively, this translated to improved fungal clearance and a shift in the immune response away from IL-1-driven and allergic hyperinflammation upon treatment of mice with Sc3458. These findings not only advance our understanding of fungal-host-pathogen interactions but also pave the way for clinical studies to validate \u003cem\u003eS. cerevisiae\u003c/em\u003e as a live biotherapeutic agent to manage fungal infection and inflammatory responses in VVC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eS. cerevisiae\u003c/b\u003e \u003cb\u003estrains and culture conditions.\u003c/b\u003e During this research, the strains utilized included \u003cem\u003eC. albicans\u003c/em\u003e SC5314\u003csup\u003e101\u003c/sup\u003e, CA3153\u003csup\u003e102\u003c/sup\u003e, and I49 (vaginal isolate). For the \u003cem\u003eS. cerevisiae\u003c/em\u003e strains, various \u003cem\u003eS. cerevisiae\u003c/em\u003e strains were employed, namely 4609 (PYCC), 6883 (DBVPG), 6886 (DBVPG), 6888 (DBVPG), 6894 (DBVPG), 3458 (NCYC), 3471 (NCYC), 3076 (NCYC), Y.00240 (NCAIM), S288C (Cold Spring Harbor Laboratory), and CNCM I-3856 (Cerevisia\u0026reg;). Before conducting all assays, individual colonies were selected from yeast extract peptone dextrose (YPD) agar plates and transferred to liquid YPD medium. Subsequently, the cultures were incubated overnight in a shaking incubator (240 rpm) at 30\u0026deg;C. The cells were harvested by centrifugation (4500 rpm, 1 min), subjected to two washes in 1 x phosphate-buffered saline (PBS), and adjusted to the desired optical density.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth evaluation.\u003c/b\u003e Growth was assessed by adjusting the optical densities of \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e cells to 0.1 and 0.5 at 600 nm, respectively. The evaluation was conducted in VSM at a pH of 4.2 (Table S2) or in RPMI 1640 medium (Gibco)\u003csup\u003e\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e,\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eC. albicans\u003c/em\u003e growth in the co-cultures was assessed at 37\u0026deg;C and 760 rpm in 96-well plates, after 6 and 24 hours, using fluorescently tagged \u003cem\u003eC. albicans\u003c/em\u003e strains. Fluorescence measurements (Excitation: 569nm, Emission: 600nm) of \u003cem\u003eC. albicans\u003c/em\u003e, tagged using the Cip10-mScarlet-IDT plasmid\u003csup\u003e\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e (CaSC5314 \u003cem\u003eACT1p\u003c/em\u003e -mScarlet, CaCA3153A \u003cem\u003eACT1p\u003c/em\u003e-mScarlet, CaI49 \u003cem\u003eACT1p\u003c/em\u003e-mScarlet), were performed using a Synergy\u0026trade; H1 microplate reader (BioTek).\u003c/p\u003e \u003cp\u003e \u003cb\u003eImaging of cultures.\u003c/b\u003e The imaging of growth was conducted according to the protocol used for the growth evaluation assay. Cocultures were visualized using a Fluoview FV1000 confocal laser scanning microscope, with the GFP fluorophore excited by a 488 nm laser and emission detected through a BA505\u0026ndash;565 bandpass filter\u003csup\u003e\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e. Five representative, randomly selected positions were imaged for each condition, and a representative image was selected.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCulturing of vaginal epithelial cells.\u003c/b\u003e To effectively mimic the vaginal epithelial layer, A-431 VECs (ACC 91) were employed. These cells, derived from vulva epidermoid carcinoma, are frequently utilized as a representative model for the vaginal niche\u003csup\u003e\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e,\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u003c/sup\u003e. Before experimentation, the cells were screened for mycoplasma contamination using a PCR mycoplasma test kit (Sigma Aldrich). A-431 VECs were cultured in RPMI 1640 medium (Gibco), supplemented with 10% fetal calf serum (FCS), and maintained at 37\u0026deg;C in a 5% CO2 atmosphere with constant humidity. During experimental assays, cells were seeded at the desired density.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdhesion to VECs.\u003c/b\u003e VECs were seeded at a density of 2 x 10\u003csup\u003e4\u003c/sup\u003e cells per well in 96-well plates and incubated at 37\u0026deg;C with 5% CO2 for two days, or until confluent. \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e cells were both adjusted to a Multiplicity of Infection (MOI) of 5. VECs were infected with \u003cem\u003eC. albicans\u003c/em\u003e cells, with or without an \u003cem\u003eS. cerevisiae\u003c/em\u003e strain (100 \u0026micro;L of 10\u003csup\u003e6\u003c/sup\u003e cells/mL in RPMI 1640 without FCS), and incubated for 90 min at 37\u0026deg;C and 5% CO2. In the assessment of \u003cem\u003eS. cerevisiae's\u003c/em\u003e potential to displace \u003cem\u003eC. albicans\u003c/em\u003e, \u003cem\u003eC. albicans\u003c/em\u003e was initially introduced alone for 90 min. Subsequently, non-adherent cells were removed by rinsing with PBS, and the \u003cem\u003eS. cerevisiae\u003c/em\u003e cells were added for an additional 90 min. For the final readout, the medium was removed, and the wells were gently rinsed with PBS to eliminate non-adherent or loosely adherent cells. To detach the adhered \u003cem\u003eC. albicans\u003c/em\u003e cells, the wells underwent trypsinization using 0.5% trypsin EDTA (Gibco), followed by sonication and dilution for a suitable colony forming units (CFU) readout on CHROMagar\u0026trade; \u003cem\u003eCandida\u003c/em\u003e (BioTrading), enabling differentiation between \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFilamentation evaluation.\u003c/b\u003e \u003cem\u003eC. albicans\u003c/em\u003e filament formation was assessed in both VSM and RPMI. The experimental setup was similar to that of the growth assay, except for adjusting the VSM pH to 5 to facilitate early filament formation. Following a 3-hour incubation at 37\u0026deg;C at 400 rpm, filament formation was quantified by manually counting filamentous cells using a Burker chamber under a Zeiss microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEpithelial damage to VECs.\u003c/b\u003e VECs were seeded as described in the adhesion assay and infected with \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eS. cerevisiae\u003c/em\u003e cells as described above, however, both at MOI 0.5, and incubated for 24 hours at 37\u0026deg;C with 5% CO2. To assess necrotic epithelial damage, the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) was measured using the Cytotoxicity Detection Kit from Roche\u003csup\u003e\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e\u003c/sup\u003e. LDH activity was quantified in the supernatant of infected VEC monolayers in 96-well plates 24 hpi. LDH from rabbit muscle (5 mg/ml, Roche) was utilized to establish a standard curve for determining LDH concentrations. The background LDH activity from uninfected VECs was subtracted from the test conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytokine release by VECs.\u003c/b\u003e Similar to the setup above, supernatants from VECs infected with \u003cem\u003eC. albicans\u003c/em\u003e, with or without the addition of \u003cem\u003eS. cerevisiae\u003c/em\u003e, were analyzed for the presence of inflammatory cytokines (IL-α, IL-8, GM-CSF). Supernatant cytokine concentrations were measured using human enzyme-linked immunosorbent assays (ELISA; R\u0026amp;D Systems) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCluster analysis.\u003c/b\u003e Cluster analysis was performed to identify patterns of similarity and the top-performing \u003cem\u003eS. cerevisiae\u003c/em\u003e strains based on their rankings across the assays. Clustering quality was assessed using the silhouette width metric, which ranges from 1 (indicating a strong fit) to -1 (indicating a poor fit). To determine the optimal number of clusters (k), K-means and K-medoids algorithms were applied, with the best clustering identified by maximizing the silhouette width\u003csup\u003e\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e. Data analysis and visualization were performed using RStudio (RStudio Team, 2020) with the R programming language.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA isolation and pooling.\u003c/b\u003e VEC were seeded in 6-well plates at 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and cultured until confluent for 2 days. Following medium removal, VECs were infected with \u003cem\u003eC. albicans\u003c/em\u003e at MOI 5, either alone or with \u003cem\u003eS. cerevisiae\u003c/em\u003e at MOI 5 (750 \u0026micro;L of 3 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL in RPMI 1640 without FCS) at 37\u0026deg;C with 5% CO2. RNA samples were collected at 6 and 24 hpi. Plates were centrifuged cool for 5 min at 2000 rpm, after which the supernatant was replaced with 500 \u0026micro;L of RLT buffer (QIAGEN), containing 1% β-mercaptoethanol. The cells were detached using a cell scraper, immediately shock-frozen in liquid nitrogen, and stored at -80\u0026deg;C until further use. For RNA extraction, the samples were thawed on ice and centrifuged for 10 min at 14000 rpm at 4\u0026deg;C. The supernatant was collected and transferred to a new microcentrifuge tube for the isolation of human RNA, while the resultant pellet was used to isolate fungal RNA. Both human and fungal RNA were isolated using the TRIzol-chloroform method, as previously described\u003csup\u003e\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u003c/sup\u003e. The RNA concentration and quality of both human and fungal RNA were assessed using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eTo ensure sufficient library depth for both human and fungal RNA, we evaluated various pooling strategies\u003csup\u003e\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u003c/sup\u003e. Based on these evaluations, fungal and human RNA samples were combined in a 1:2 ratio by concentration for library preparation and sequencing, aiming to achieve adequate sequencing depth for comprehensive differential gene expression analysis. Before pooling, we measured cross-mapping rates by aligning reads from strictly human and fungal samples to respective fungal and human genomes, resulting in no cross-mapped reads between the three species.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq library preparation and sequencing.\u003c/b\u003e Bulk RNA sequencing (RNA-seq) libraries were prepared by Novogene (Directional mRNA enrichment libraries) and subjected to NovaSeq paired-end sequencing (2 \u0026times; 150 base pairs). Sequencing depth and read length were optimized to ensure comprehensive coverage and reliable differential gene expression analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-seq pipeline.\u003c/b\u003e Paired-end FASTQ files were obtained from Novogene for subsequent analysis. Initial quality control of the raw sequencing data was conducted using FastQC and MultiQC\u003csup\u003e\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e,\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e\u003c/sup\u003e. For read alignment and quantification, we employed the splice junction-sensitive aligner STAR\u003csup\u003e\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e\u003c/sup\u003e. Given that the samples included RNA from both host and pathogen, we performed read mapping against a concatenated reference comprising both the human and \u003cem\u003eC. albicans\u003c/em\u003e genomes. Human reads were aligned to the primary genome assembly GRCh38, with annotations derived from NCBI (last accessed May 2, 2024). The \u003cem\u003eC. albicans\u003c/em\u003e SC5314 genome assembly ASM18296v3 and associated annotations were sourced from NCBI (last accessed May 2, 2024). BEDtools (intersectBed) and Picard were used to remove rRNA reads and optical duplicates from the mapped reads\u003csup\u003e\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e,\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e\u003c/sup\u003e. Samtools was used for indexing and generating summary statistics\u003csup\u003e\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e\u003c/sup\u003e. htseq-count was used to count the number of genes within each BAM file\u003csup\u003e\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e\u003c/sup\u003e. Coverage was evaluated using deepTools (bamCoverage) and BEDtools\u003csup\u003e\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e,\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e\u003c/sup\u003e. Sequence alignment was assessed using BLAST\u003csup\u003e\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eData analysis and visualization were performed using RStudio (RStudio Team, 2020) with the R programming language. Differential gene expression analysis was performed using the Bioconductor package edgeR v.3.34.0\u003csup\u003e121\u003c/sup\u003e. Read counts were obtained from STAR mapping. For human samples and each fungal species, we compared time point 0 with other time points throughout the infection course using the glmQLFTest function from edgeR. We used filterByExpr to filter out low-count genes. The dispersions were estimated using the estimateDisp function, and differential expression was assessed by applying a contrast matrix with glmQLFTest. Genes with a log2(fold change)\u0026thinsp;\u0026gt;\u0026thinsp;1.0 and an adjusted P (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered differentially expressed. To visualize the data, we performed Principal Component Analysis (PCA) on normalized counts and created plots using ggbiplot. Differentially expressed gene results were visualized with scatter and volcano plots. Gene ontology enrichment was performed using ShinyGO\u003csup\u003e\u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e\u003c/sup\u003e. Following this, the significantly enriched GO terms were analyzed using REVIGO85, applying a similarity threshold of Small (0.5)\u003csup\u003e\u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e\u003c/sup\u003e. All code and scripts written for this paper can be accessed on the following online repository: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/dania-ab/VVC\u003c/span\u003e\u003cspan address=\"https://github.com/dania-ab/VVC\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm biomass.\u003c/b\u003e \u003cem\u003eC. albicans\u003c/em\u003e biomass formation on VECs was assessed by measuring the fluorescence emitted by fluorescently tagged \u003cem\u003eC. albicans\u003c/em\u003e strains. The experimental procedure followed the adhesion assay protocol: VECs were seeded and cultured until confluent, then infected with \u003cem\u003eC. albicans\u003c/em\u003e, with or without \u003cem\u003eS. cerevisiae\u003c/em\u003e. At 24 and 48 hpi, the medium was aspirated, and the wells were gently washed with PBS to remove non-adherent or loosely attached cells. Subsequently, biomass quantification was performed using fluorescence measurements on a Synergy\u0026trade; H1 microplate reader (BioTek), with excitation set to 569 nm and emission to 590 nm\u003csup\u003e\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e. For experiments involving transwells, the cells were physically separated using a ThinCert\u0026trade; Cell Culture insert (Greiner Bio-one) with a 0.4 \u0026micro;m pore size. For experiments involving heat-inactivated \u003cem\u003eS. cerevisiae\u003c/em\u003e cells, inactivation was performed by incubating the cells in 1X PBS at 65\u0026deg;C for 30 minutes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm distribution.\u003c/b\u003e Biofilms were established using the same protocol as the biofilm biomass assay. At 24 hpi, the supernatant was collected, and the wells were gently washed with PBS to remove non-adherent or loosely attached cells. To detach the adhered \u003cem\u003eC. albicans\u003c/em\u003e cells, the wells were treated with 0.5% trypsin-EDTA (Gibco), followed by water bath sonication. The resulting cell suspension was then diluted to obtain an appropriate CFU count on CHROMagar\u0026trade; \u003cem\u003eCandida\u003c/em\u003e (BioTrading).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiofilm structure.\u003c/b\u003e Biofilms were established following the same protocol as the biofilm biomass assay but were grown on IBIDI plates. Imaging of the biofilms was performed using a Fluoview FV1000 confocal laser scanning microscope. The mScarlet fluorophore was excited with a 559 nm laser, and emission was captured through a BA575\u0026ndash;675 bandpass filter\u003csup\u003e\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e. A series of images were acquired at 4.51 \u0026micro;m intervals along the z-axis, allowing for a three-dimensional reconstruction of the biofilms from the start to the end of the fluorescent signal. The z-stack images were subsequently exported to the Napari interface for further analysis\u003csup\u003e\u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of hMDMs.\u003c/b\u003e Buffy coats obtained from healthy volunteers underwent Histopaque-1077 (Sigma-Aldrich) density centrifugation to isolate human peripheral blood mononuclear cells (hPBMC). CD14-positive monocytes were subsequently isolated using magnetic automated cell sorting (autoMACs; MiltenyiBiotec). These cells were then differentiated into human monocyte-derived macrophages (hMDMs) by seeding 1.7 x 10\u003csup\u003e7\u003c/sup\u003e cells in a 175 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e cell culture flask containing RPMI 1640 medium with 2 mM \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-glutamine (Thermo Fisher Scientific), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Bio\u0026amp;SELL), and 50 ng/mL recombinant human M-CSF (ImmunoTools). The cells were incubated for seven days at 37\u0026deg;C and 5% CO2 with a medium exchange on day 5. The adherent hMDMs were detached using 50 mM EDTA in PBS, then seeded in 96-well plates at a final concentration of 4 x 10\u003csup\u003e4\u003c/sup\u003e hMDMs/well in RPMI\u0026thinsp;+\u0026thinsp;FBS\u0026thinsp;+\u0026thinsp;M-CSF, and incubated overnight. The macrophage infection experiments were conducted in serum-free RPMI medium.\u003c/p\u003e \u003cp\u003e \u003cb\u003ehMDMs stimulation.\u003c/b\u003e To assess the stimulation of hMDMs, the release of IL-1β was measured, as it serves as an indicator of NLRP3 activation\u003csup\u003e\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e\u003c/sup\u003e. hMDMs were seeded at a density of 4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well, as previously described. Before infection, the phagocytes were primed by incubating them with 50 ng/mL of lipopolysaccharide (LPS) (Sigma Aldrich) for 2 hours. Subsequently, the hMDMs were infected with \u003cem\u003eC. albicans\u003c/em\u003e at MOI 0.5, with or without \u003cem\u003eS. cerevisiae\u003c/em\u003e at MOI 0.5, for 24 hours at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After the designated incubation period, the supernatant from the infected hMDMs was collected by centrifuging the plates at 250 x g for 5 min. Supernatant IL-1β concentrations were measured using human enzyme-linked immunosorbent assays (ELISA; R\u0026amp;D Systems) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation of human neutrophils.\u003c/b\u003e The isolation of primary human neutrophils followed the procedures outlined by Gresnigt et al.\u003csup\u003e\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e\u003c/sup\u003e. Initially, human peripheral blood mononuclear cells were separated from granulocytes and erythrocytes using density gradient centrifugation with Histopaque-1077 (Sigma-Aldrich) in a sterile 50 mL falcon. Neutrophils were subsequently extracted from the erythrocyte/granulocyte fraction by hypotonic lysis with a buffer containing 155 mM NH\u003csub\u003e4\u003c/sub\u003eCl and 10 mM KHCO\u003csub\u003e3\u003c/sub\u003e. Following two washes in 1 x PBS, the neutrophils were resuspended in RPMI medium and adapted to the required concentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil stimulation: IL-8 release.\u003c/b\u003e To assess neutrophil stimulation in response to \u003cem\u003eC. albicans\u003c/em\u003e and the potential influence of \u003cem\u003eS. cerevisiae\u003c/em\u003e, neutrophils were treated with supernatants. These supernatants were diluted 1:2 in RPMI and collected from VECs exposed to \u003cem\u003eC. albicans\u003c/em\u003e, with or without \u003cem\u003eS. cerevisiae\u003c/em\u003e, for 24 hours. This process was carried out as previously described. Freshly isolated neutrophils were seeded at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e neutrophils/well in a 96-well plate and following a 3-hour stimulation period at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, supernatants from the neutrophils were collected. The concentration of IL-8, an indicator of neutrophil activation, was then measured in these supernatants using an ELISA assay (R\u0026amp;D Systems) following the manufacturer\u0026rsquo;s instructions\u003csup\u003e\u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil activation: ROS production.\u003c/b\u003e Neutrophil activation in response to \u003cem\u003eC. albicans\u003c/em\u003e, with and without \u003cem\u003eS. cerevisiae\u003c/em\u003e, was evaluated using ROS production in an oxidative burst assay. To quantify the total ROS, a luminol-enhanced chemiluminescence method was performed. Freshly isolated neutrophils were seeded at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e neutrophils/well as mentioned above. Neutrophils were then incubated for 30 min at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, to let them attach. Subsequently, the neutrophils were stimulated for 1 hour 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e with undiluted supernatants from VECs exposed to \u003cem\u003eC. albicans\u003c/em\u003e, with and without \u003cem\u003eS. cerevisiae\u003c/em\u003e following the previously described protocol. After incubation, 50 \u0026micro;L of zymosan (at a concentration of 150 \u0026micro;g/mL) was added to further stimulate the fungal immune response and enhance ROS production\u003csup\u003e\u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e\u003c/sup\u003e. Following stimulation, 50 \u0026micro;l of RPMI1640 containing 200 \u0026micro;M luminol (Sigma Aldrich) and 16 U HRP (Sigma Aldrich) for total ROS detection was added. Chemiluminescence was measured every 2.5 minutes for 2.5 hours using a Tecan Infinite M200 microplate reader. The area under the curve was calculated using GraphPad Prism 10.4.3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil stimulation: Activation-associated cell surface markers.\u003c/b\u003e Further assessment of neutrophil stimulation involved analyzing the regulation of neutrophil activation markers using flow cytometry. The selection of activation markers was based on granulocyte responses to fungal pathogens or related stimuli\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Neutrophils were seeded in a round-bottom 96-well plate at a density of 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and stimulated with undiluted supernatants from VECs exposed 24 h to \u003cem\u003eC. albicans\u003c/em\u003e, with and without \u003cem\u003eS. cerevisiae\u003c/em\u003e, following the previously described protocol. After a 3 h stimulation period at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, supernatants were removed, and the neutrophils were washed with flow cytometry buffer (PBS, 2% FCS). To minimize nonspecific staining, neutrophils were first incubated with Fc-block Human TruStain FcX (BioLegend). Subsequently, a mixture of fluorophore-linked antibodies targeting surface molecules indicative of activation status was added: CD15-APC-Fire750 (W6D3), CD11b-BV421 (ICR44), CD16-PerCP-Cy5.5 (3G8), CD35-FITC (E11), CD62L-AlexaFluor647 (DREG-56), CD66b-PE (G10F5), and CXCR2 (5E8, all from BioLegend). Fixable Viability Dye eFluor506 (Invitrogen) was added to exclude non-viable cells for 20 min at 8\u0026deg;C. Subsequently, cells were washed with flow cytometry buffer, filtered through a 70 \u0026micro;m mesh, and then analyzed using a FACSVerse Cell Analyzer flow cytometer (BD Biosciences). Gating strategies for granulocyte populations and activation markers followed the approach described by Valentine et al.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Data analysis was carried out using FlowJo v.10.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil chemotaxis.\u003c/b\u003e Neutrophil chemotaxis was performed as described by Valentine et al.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Briefly, primary human neutrophils were stained with Cytopainter Green (2 \u0026micro;L in 1 \u0026times; 10⁶ neutrophils suspended in RPMI), incubated for 10 min at room temperature in the dark. Cells were then washed with Hank\u0026rsquo;s Balanced Salt Solution containing 20 mM HEPES (pH 7), centrifuged (10 min, 300 \u0026times; g), and resuspended in endothelial cell medium (ECM, PromoCell) at 5 \u0026times; 10⁵ cells/mL. Human umbilical vein endothelial cells (HUVECs) were cultured for 72 h in 150-cm\u0026sup2; flasks, harvested, and seeded into 3 \u0026micro;m transwell inserts at 2 \u0026times; 10⁴ cells/insert. After 48 h, inserts were transferred to 24-well plates containing confluent VECs, seeded two days earlier at 1 \u0026times; 10⁵ cells/well. Medium in the inserts was refreshed (200 \u0026micro;L), and VECs were infected with \u003cem\u003eC. albicans\u003c/em\u003e (1 \u0026times; 10⁵ cells, MOI 0.5) in the presence or absence of \u003cem\u003eS. cerevisiae\u003c/em\u003e (1 \u0026times; 10⁵ cells, MOI 0.5) in a total volume of 600 \u0026micro;L. After 18 h, 200 \u0026micro;L of stained neutrophils were added to the inserts.. Plates were incubated for 2 h at 37\u0026deg;C and 5% CO₂. Images were acquired using an IncuCyte SX5 live-cell imaging system (Sartorius). Thresholds for fluorescence intensity and area size ensured accurate segmentation and quantification. Image analysis was performed using Incucyte 2023A Rev2 software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil death and NETosis evaluation.\u003c/b\u003e To assess the influence of \u003cem\u003eS. cerevisiae\u003c/em\u003e on \u003cem\u003eC. albicans\u003c/em\u003e-induced neutrophil responses, co-cultures were established to evaluate NETosis and neutrophil cell death. Neutrophils (1 \u0026times; 10⁵ cells per well) were pre-stained with Sytox Green (4 \u0026times; 10⁻⁵ \u0026micro;L/mL RPMI) to monitor NETosis and cell death, while \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e were added at an MOI of 0.5. Staining was performed in the dark at 37\u0026deg;C with 5% CO₂ for 15 min prior to co-culture. Co-cultures were imaged in an Incucyte SX5 (Sartorius) at 37\u0026deg;C with 5% CO₂. Negative controls were left unstimulated, positive controls were stimulated with 20 nM PMA. NETosis and cell death were monitored in real-time using green fluorescence imaging. Images were captured from two regions per well every 20\u0026ndash;30 min for 12 h at \u0026times;20 magnification, with an exposure time of 100 ms. Representative images and images of unstimulated and PMA-induced NETosis conditions were used to train the Incucyte Basic Software for analysis. The Top-Hat method was applied for background correction, and the edge-split tool was used to distinguish closely spaced objects. Thresholds for fluorescence intensity and area size ensured accurate segmentation and quantification. Image analysis was performed using Incucyte 2023A Rev2 software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil-mediated\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003ekilling and growth.\u003c/b\u003e To evaluate the effects of \u003cem\u003eS. cerevisiae\u003c/em\u003e on neutrophil-mediated \u003cem\u003eC. albicans\u003c/em\u003e growth impairment, we used a setup similar to that described above, incorporating mScarlet-tagged \u003cem\u003eC. albicans\u003c/em\u003e. Fungal overgrowth was monitored in the orange fluorescence channel using the Incucyte live-cell imaging system, with an exposure time of 300 ms. The Adaptive method was applied for image analysis to account for variations in fluorescence intensity and object morphology during fungal growth. Data were analyzed using Incucyte 2023A Rev2 software. To assess the influence of \u003cem\u003eS. cerevisiae\u003c/em\u003e on neutrophil-mediated \u003cem\u003eC. albicans\u003c/em\u003e killing, a similar co-culture system was used, except that unstained neutrophils and \u003cem\u003eC. albicans\u003c/em\u003e were included. Killing assays were performed at 1 and 3 hpi. After co-culture, neutrophils were lysed with 4% Triton X-100, and samples were scraped, sonicated, serially diluted, and plated on CHROMagar\u0026trade; \u003cem\u003eCandida\u003c/em\u003e (BioTrading) to differentiate \u003cem\u003eC. albicans\u003c/em\u003e from \u003cem\u003eS. cerevisiae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutrophil-mediated\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003ebiofilm eradication.\u003c/b\u003e To evaluate whether the biofilm-inhibiting effects of \u003cem\u003eS. cerevisiae\u003c/em\u003e enhance \u003cem\u003eC. albicans\u003c/em\u003e clearance by neutrophils, we assessed neutrophil-mediated killing in co-culture with \u003cem\u003eC. albicans\u003c/em\u003e biofilms. Following the biofilm biomass quantification protocol, co-cultures were incubated for 24 h, washed to remove non-adherent cells, and fluorescent \u003cem\u003eC. albicans\u003c/em\u003e values were measured to normalize the data, accounting for reduced biofilm biomass due to \u003cem\u003eS. cerevisiae\u003c/em\u003e. 5 \u0026times; 10⁵ neutrophils per well were added and incubated with the biofilms for 3 hours at 37\u0026deg;C with 5% CO₂ to allow for \u003cem\u003eC. albicans\u003c/em\u003e killing. After co-culture, neutrophils were lysed with 4% Triton X-100, and biofilms were scraped, sonicated, serially diluted, and plated on CHROMagar\u0026trade; \u003cem\u003eCandida\u003c/em\u003e (BioTrading) to differentiate \u003cem\u003eC. albicans\u003c/em\u003e colonies from \u003cem\u003eS. cerevisiae\u003c/em\u003e. This method enabled precise quantification of \u003cem\u003eC. albicans\u003c/em\u003e survival and biofilm clearance.\u003c/p\u003e \u003cp\u003e\u003cb\u003eMurine model of VVC.\u003c/b\u003e Female BALB/cJ mice 8 weeks old were obtained from Charles River and used for the study. To induce a pseudoestrous state, mice received a subcutaneous injection of 0.1 mg β-estradiol 17-valerate (Sigma), dissolved in 100 \u0026micro;L of sesame oil, 72 hours before inoculation. Estrogen administration was continued weekly throughout the experiment\u003csup\u003e\u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e\u003c/sup\u003e. To establish infection, estrogen-treated mice were sedated and inoculated with 10 \u0026micro;L of 10\u003csup\u003e7\u003c/sup\u003e bioluminescent (BLI) \u003cem\u003eC. albicans\u003c/em\u003e cells/mL, suspended in 1 \u0026times; PBS. The inoculum was administered intravaginally via pipetting. To minimize leakage and enhance fungal retention, mice were positioned with their hindquarters elevated for 20 minutes post-inoculation. Following a 24-hour incubation period to allow for infection establishment, mice received intravaginal treatment with either 10 \u0026micro;L of 10\u003csup\u003e8\u003c/sup\u003e\u003cem\u003eS. cerevisiae\u003c/em\u003e 3458 cells/mL or 1 \u0026times; PBS (control), similar to \u003cem\u003eC. albicans\u003c/em\u003e infection. Treatments were initiated one day post-inoculation and continued daily. The experimental protocols were reviewed and approved by the Ethical Committee for Animal Experimentation of KU Leuven under approval number Bio-157/2024.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMonitoring\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003evaginal load.\u003c/b\u003e Fungal burden was assessed on days 2, 4, 6, 8, and 10 post-infection using bioluminescence imaging (BLI). Mice were anesthetized with 2.5\u0026ndash;3.5% (v/v) isoflurane gas, followed by maintenance at 1.5\u0026ndash;2% isoflurane in oxygen (Piramal Critical Care). D-luciferin (1.1665 mg/mL, 10 \u0026micro;L) was administered intravaginally. After anesthesia induction, mice were positioned supine in an IVIS Spectrum System (Perkin-Elmer, Hopkinton, MA, USA). Imaging began 5 minutes post-injection, capturing five consecutive images using the following parameters: 30-second exposure time, medium binning, F/stop of 1, and subject height of 1.5 cm. Bioluminescence signal quantification was performed using Living Image Software (version 4.7.3, Perkin-Elmer). Total photon flux (photons/second) was measured within a region of interest (ROI) of 2.8 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, covering the vaginal area. The maximum signal from consecutive acquisitions was used for further analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMonitoring inflammatory state.\u003c/b\u003e To assess the presence of inflammatory markers, vaginal lavages were performed. Each lavage was conducted using 200 \u0026micro;L of 1 \u0026times; PBS, administered in four consecutive volumes of 50 \u0026micro;L each. Following collection, the lavage samples were centrifuged at 1200 rpm for 10 minutes at 4\u0026deg;C to separate soluble components from immune cells. The resulting supernatants were collected and subjected to quantitative analysis using a Luminex Mouse Discovery Assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry.\u003c/b\u003e Flow cytometry was performed to assess the presence of macrophages, neutrophils and eosinophils in vaginal lavage fluid. Lavage samples were centrifuged at 1,200 rpm for 10 min at 4\u0026deg;C to separate soluble components and immune cells. The resulting cell pellet was washed with ice-cold 1 x PBS and incubated with Fc-receptor blocking antibodies (anti-CD16/CD32 Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 minutes at room temperature in the dark (\u003cb\u003eTable S3\u003c/b\u003e). Simultaneously, a Zombie Aqua\u0026trade; viability dye (BioLegend, USA) was applied to discriminate live from dead cells. Following incubation, the cells were washed with cold 1 x PBS supplemented with 2% FCS-2mM EDTA and stained with a surface marker antibody cocktail (\u003cb\u003eTable S3\u003c/b\u003e) at 4\u0026deg;C for 30 minutes. After staining, cells were washed with cold PBS containing 2% FBS and resuspended in PBS supplemented with 2% v/v FBS and 2 mM EDTA to prevent cell aggregation. Samples were maintained at 4\u0026deg;C until analysis. Flow cytometry was conducted using a BD LSRFortessa\u0026trade; X-20 (BD Biosciences) and data analysis was carried out using FlowJo software (BD Biosciences, v10). The specific gating strategies are depicted in \u003cb\u003eFig. S10\u003c/b\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Marisa Valentine and Sophie Austermeier for their technical assistance with macrophage and neutrophil functional assays. We also thank Wouter Van Genechten for his contributions to confocal microscopy imaging and the development of custom scripts for quantitative analysis of biofilm thickness. Nele Berghmans is acknowledged for her support with flow cytometry acquisition and analysis.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFWO-funded SBO project DeVEnIR S006424N (P.V.D., M.S.G., S.L.)\u003c/p\u003e\n\u003cp\u003eFWO research community on Biofilms (W000921N) (P.V.D.)\u003c/p\u003e\n\u003cp\u003eFWO PhD fellowship 1SD8622N (M.S.)\u003c/p\u003e\n\u003cp\u003eFlanders Innovation \u0026amp; Entrepreneurship VLAIO HBC.2021.824 (S.B.)\u003c/p\u003e\n\u003cp\u003eFWO-funded SBO project DeVEnIR S006424N (P.V., K.O.C., I.P.)\u003c/p\u003e\n\u003cp\u003eMicroverse visiting scientist grant (M.S.)\u003c/p\u003e\n\u003cp\u003eExploration Grant of the Boehringer Ingelheim Foundation (BIS) (M.S.G., A.D.)\u003c/p\u003e\n\u003cp\u003eFree State of Thuringia and European Union \u0026ndash; Project-ID 2023 FGI 0004. \u0026quot;A Live broadcast of the interactions between host and fungal pathogens\u0026quot; (M.S.G, A.D.)\u003c/p\u003e\n\u003cp\u003eGerman Research Foundation (Deutsche Forschungsgemeinschaft - DFG) Emmy Noether Program 434385622 / GR 5617/1-1 (M.S.G.)\u003c/p\u003e\n\u003cp\u003eRega Foundation (M.G.)\u003c/p\u003e\n\u003cp\u003eEmmy Noether Programme (D.E.M)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: M.S., P.V.D., M.S.G., L.D., S.L.\u003c/p\u003e\n\u003cp\u003eMethodology: M.S., D.E.A., S.B., D.E.M., G.V.V., A.R.S., P.V., M.S.G., A.D., L.D., M.G.\u003c/p\u003e\n\u003cp\u003eInvestigation: M.S., D.E.A., N.V., S.B., K.O.C., O.V.G., I.P., A.D.\u003c/p\u003e\n\u003cp\u003eSoftware: M.S., D.E.A.\u003c/p\u003e\n\u003cp\u003eVisualization: M.S., D.E.A., M.S.G., A.D.\u003c/p\u003e\n\u003cp\u003eFunding acquisition: M.S., P.V.D., S.L., L.D., M.S.G.\u003c/p\u003e\n\u003cp\u003eProject administration: M.S., P.V.D.\u003c/p\u003e\n\u003cp\u003eSupervision: P.V.D, M.S.G, S.L., L.D.\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: M.S., M.S.G., A.D., D.E.A.\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Review \u0026amp; Editing: M.S., D.E.A., L.D., P.V., I.P., A.D., S.L., M.S.G., M.G., P.V.D.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIlkit, M. \u0026amp; Guzel, A. 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Effects of Reproductive Hormones on Experimental Vaginal Candidiasis. \u003cem\u003eInfection and Immunity\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 651-657, doi:10.1128/iai.68.2.651-657.2000 (2000).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Candida albicans, biofilm formation, neutrophil activation, bulk RNA-seq, epithelial infection, VVC mice model","lastPublishedDoi":"10.21203/rs.3.rs-6795973/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6795973/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVulvovaginal candidiasis (VVC) affects millions globally and is characterized by multifactorial immunopathology, with \u003cem\u003eCandida albicans\u003c/em\u003e virulence driving disease progression through epithelial tissue damage and neutrophil hyperactivation and dysfunction contributing to disease severity. Here, we explored the multifaceted nature of \u003cem\u003eS. cerevisiae\u003c/em\u003e as a live-biotherapeutic to attenuate \u003cem\u003eC. albicans\u003c/em\u003e virulence and modulate host immune responses during VVC. We identified an \u003cem\u003eS. cerevisiae\u003c/em\u003e isolate that targets multiple aspects of \u003cem\u003eC. albicans\u003c/em\u003e virulence, including fungal proliferation, adhesion, and hyphal morphogenesis, collectively impairing biofilm formation and disrupting pathogenic potential. These effects were linked to transcriptional reprogramming in \u003cem\u003eC. albicans\u003c/em\u003e, marked by metabolic stress and downregulation of virulence- and biofilm-related genes. Additionally, \u003cem\u003eS. cerevisiae\u003c/em\u003e reduced inflammatory responses and neutrophil hyperactivation, while preserving neutrophil antimicrobial functionality. Collectively, this translated to an improved control of infection and dampened VVC-associated hyperinflammation in a murine VVC model. These findings position \u003cem\u003eS. cerevisiae\u003c/em\u003e as a promising live biotherapeutic to mitigate \u003cem\u003eC. albicans\u003c/em\u003e infection and alleviate hyperinflammation in VVC. Further clinical validation in human cohorts is essential to confirm its therapeutic efficacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOne Sentence Summary: \u003c/strong\u003e\u003cem\u003eS. cerevisiae\u003c/em\u003e attenuates \u003cem\u003eC. albicans\u003c/em\u003e virulence and inflammation, offering therapeutic potential for vulvovaginal candidiasis.\u003c/p\u003e","manuscriptTitle":"Saccharomyces cerevisiae reduces vulvovaginal candidiasis severity through modulation of fungal pathogenicity and inflammatory responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 08:16:30","doi":"10.21203/rs.3.rs-6795973/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ef3afd27-487d-43e0-ba25-a84211eaf1b9","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":50165342,"name":"Health sciences/Diseases/Infectious diseases/Fungal infection"},{"id":50165343,"name":"Biological sciences/Microbiology/Fungi/Fungal host response"},{"id":50165344,"name":"Biological sciences/Microbiology/Cellular microbiology"}],"tags":[],"updatedAt":"2025-06-17T08:16:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 08:16:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6795973","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6795973","identity":"rs-6795973","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}