Elucidation of the secretory crosstalk between the pathogens Candida albicans and Aspergillus fumigatus via multimodal metabolomics

Elucidation of the secretory crosstalk between the pathogens Candida albicans and Aspergillus fumigatus via multimodal metabolomics | 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 Elucidation of the secretory crosstalk between the pathogens Candida albicans and Aspergillus fumigatus via multimodal metabolomics Sophie Tonneau, Denis Ispan, Gyuntae Bae, Jannik Sprengel, Frederic Dalle, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9059703/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Candida albicans (C. albicans) and Aspergillus fumigatus ( A. fumigatus) are major opportunistic species, classified by the World Health Organization(WHO) in 2022 as critical priority pathogen due to their high morbidity and mortality rates. Despite the prevalence of Candida-Aspergillus coinfections in organ transplant recipients and patients with hematologic malignancies, mechanisms underlying their biological interplay remain poorly understood. Using a combinatory metabolomics approach of targeted proton Nuclear Magnetic Resonance spectroscopy and untargeted metabolomics by trapped ion mobility spectrometry time-of-flight mass spectrometry, we investigated metabolic interactions between these pathogens and annotated a total of 176 compounds. We highlighted ten A. fumigatus mycotoxins, among them, sphingofungin B and D and spirotryprostatin A, decreased in the presence of C. albicans secretomes, indicating a potential inhibitory effect. Notably, C. albicans cultures exposed to conidia secretome formed a distinct metabolic cluster. In coculture, an inhibition zone with fragmented A. fumigatus hyphae was observed, suggesting hyphal damage. Despite this, both species could grow together, highlighting their capacity of cohabitation albeit displaying antagonistic metabolic interactions. The observed inhibition zone and mycotoxin modulation suggest a competitive, yet non-lethal interaction characterized by unique metabolomic chemo-sensing. Importantly, the production of tissue-damaging mycotoxins such as sphingofungin B and D, TR-2 in A. fumigatus - C. albicans coinfection may predispose infected sites to polymicrobial invasion, exacerbating disease severity and persistence, impairing treatment efficacy and worsening patient prognosis. Health sciences/Diseases Biological sciences/Microbiology Candida albicans Aspergillus fumigatus fungal interaction metabolic exchange profiling H-1-NMR timsTOF LC-MS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION The incidence of fungal superinfections is rising in both immunocompromised and immunocompetent patients. Among the fungal pathogens implicated in superinfections, A. fumigatus and C. albicans are the most prevalent worldwide and the primary causative agents of invasive fungal infections, particularly pulmonary infections and bloodstream infections 1 , 2 . While the clinical burden of A. fumigatus and C. albicans coinfection is substantial, it remains often underrecognized. Both pathogens co-occur in patients with cystic fibrosis, invasive pulmonary aspergillosis complicated by candidiasis, or pleural infections 3 , 4 . For example, in cystic fibrosis patients, fungal prevalence reaches 45.7% for A. fumigatus and 75.5% for C. albicans , with A. fumigatus coinfection occurring in 60% of cases alongside P . aeruginosa 3 , 5 , 6 . Similarly, among COVID-19 patients, fungal infections are predominantly caused by A. fumigatus (3.71%) 7 and C. albicans (2.39%) 8–11 . Critically, C. albicans colonizes over 50% of patients asymptomatically 12 creating a reservoir for opportunistic invasion when immune defences are compromised. Dual fungal infections pose two interconnected threats that compromise patient survival. First, polymicrobial infections fundamentally alter disease dynamics compared to single-species infections 1 , 12 . The presence of one species may enhance the virulence, abundance, or persistence of others, establishing a self-reinforcing pathogenic cycle 13 – 15 . Second, with pathogens with high colonization potential, such as A. fumigatus and C. albicans , there is a risk of persistent infections that are difficult to eradicate 1 , 12 , 16 . Together, these factors create a silent clinical crisis: two pathogens coexist and synergistically worsen patient prognosis while presenting as a single infection. Despite the clinical urgency of dual fungal infections, fungal–fungal interactions remain strikingly under-explored compared to other polymicrobial systems 17 . Bacteria–bacteria, bacteria–fungi and viruses-fungi pairs in both in vitro and in vivo models 13 , 18 . For instance, P. aeruginosa demonstrated complex interactions with both study fungi: it can both inhibit and promote A. fumigatus growth in cystic fibrosis biofilm, while producing yeast-toxic pigments against C. albicans 2 , 5 , 19 – 23 . Conversely, C. albicans colonization predisposes patients to P. aeruginosa pneumonia, illustrating the clinical significance of these interactions 2 , 19 , 24 . An example of bacterial-fungal synergism is the capacity of Staphylococcus aureus and E. coli to enhance C. albicans adhesion to mucosal surfaces 2 , 25 . Additionally, viral infections, particularly COVID-19, predispose patients to A. fumigatus superinfection through epithelial damage, facilitating conidia internalization into cells 26 , 27 . In contrast, fungal–fungal interactions, particularly between opportunistic pathogens, remain largely unexplored. While one study reported fungistatic effects of C. albicans against A. fumigatus in vitro , the underlying mechanisms were not elucidated 28 . This knowledge gap is clinically significant, as polymicrobial infections involving fungal agents are challenging to diagnose and treat, often resulting in worse patient outcomes and increased mortality rates. Given their high clinical relevance and frequent co-occurrence, understanding the crosstalk between A. fumigatus and C. albicans is essential for elucidating the dynamics of coinfection and the factors enabling fungal persistence. Here, we investigated the interactions between A. fumigatus and C. albicans in vitro using enriched cultures experiments designed to mimic dual fungal infection. To achieve this aim, we developed a secretome-metabolite exchange analysis approach using combinatory multimodal metabolomics analysis by targeted proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) and untargeted liquid chromatography (LC) trapped ion mobility spectrometry time-of-flight mass spectrometry (timsTOF LC-MS) using large CCS (collision cross section) libraries for maximum annotation coverage. To explore the role of different morphological forms in metabolite production, we studied the metabolic interaction between whole A. fumigatus culture with yeast or hyphae secretomes as well as C. albicans culture exposed to A. fumigatus secretome (conidia or hyphae). Using this multimodal targeted and untargeted approach of two state of the art metabolomics technologies enabled us to identify antagonistic behaviours and potential mutualistic mechanisms that could explain the severity and persistence of dual fungal infections in vulnerable patient populations, paving the ground for improved future target discovery and treatments. RESULTS Coculture of A. fumigatus and C. albicans shows interaction at the hyphal front . To characterize secretory metabolic interactions between A. fumigatus and C. albicans , we first performed enrichment culture experiments using secretomes from different fungal morphotypes (yeast, conidia and hyphae) added to the whole fungal culture and analysed the samples by both 1 H-NMR and timsTOF LC-MS. Our multimodal metabolomics approach could highlight differential metabolite signatures between enriched culture and culture control conditions. Figure 1 a shows the workflow for the analysis of metabolites in enriched cultures: A. fumigatus and C. albicans cultures supplemented with secretomes were grown during 5 days at 25°C. The monoculture control (containing only A. fumigatus or only C. albicans) were grown under the same conditions. Enriched cultures and monoculture were extracted with organic solvents for H¹-NMR and timsTOF LC-MS measurements. Figure 2 : A. fumigatus conidia secretome leads to shift in metabolites distribution. a Discriminant analysis score plot of metabolites obtained from pure culture A. fumigatus (red circles) and C. albicans (light blue circles) and enriched cultures: A. fumigatus supplemented with hyphae secretome (green circles), A. fumigatus with yeast secretome (purples circles), C. albicans with hyphae secretome ( A. fumigatus ) (pink circles) and C. albicans with conidia secretome (yellow circles) after 5 days incubation at 25°C. Coloured ellipses represent the 95% confidence regions of each clustering. b Clustering analysis of the different metabolites in each group. Right column of the heatmap: 27 significant metabolites identified with ANOVA test (p < 0.01). Bottom of the heatmap: C. albicans culture control in 3 replicates on the left vs. enriched cultures with A. fumigatus hyphae (4 replicates) and enriched culture with conidia secretome (3 replicates) on the right. 1 H-NMR and timsTOF LC-MS data were combined and normalized with log transformation for the statistical analysis. H¹-NMR and timsTOF LC-MS highlight the impact of A. fumigatus conidia on C. albicans metabolic activity . H¹-NMR and timsTOF LC-MS analysis were conducted to explore the metabolic interactions between C. albicans and A. fumigatus after 5 days of enriched cultures compared to the monoculture. Metabolites of interest are summarized in Table 1 ( C. albicans metabolites linked to virulence), Table 2 ( A. fumigatus mycotoxins) and Table 3 (metabolites common to both fungi) and their known biological functions described. To determine the degree of difference between the metabolite profile observed during enriched culture and monoculture, Principal Component Analysis (PCA) scores were generated based on combined H¹-NMR and timsTOF LC-MS measurements (Fig. 2a ). PCA was applied to all data sets: A. fumigatus and C. albicans monocultures and enriched culture. PCA scores resulted in three distinct clusters, where the C. albicans culture enriched with conidia secretome was separated from the other groups. There was no difference between the C. albicans monoculture and the C. albicans culture supplemented with A. fumigatus hyphae. A. fumigatus monoculture, A. fumigatus culture supplemented with C. albicans hyphae or yeast were grouped together (Fig. 2a). Secretome from conidia seems to induce a unique metabolome profile on C. albicans culture compared to the other conditions. Since our PCA analysis revealed a distinct clustering of the enriched culture with conidia secretome compared to the other conditions, we next generated a metabolic heatmap to visualize metabolite-level differences driving this separation. The metabolic heatmap generated 27 significant metabolites through ANOVA analysis (p < 0.01) from combined H¹-NMR and timsTOF LC-MS data sets (Fig. 2b). The metabolite distribution in the C. albicans culture control remained unchanged when A. fumigatus hyphae secretome was added. Among the 27 significant metabolites identified, 13 metabolites were increased in both conditions (control and C. albicans enriched culture with A. fumigatus hyphae secretome): 5-ethyl-2.4-dimethyloxazole, n-isopropyl-l-glutamine, lactose, lactate, n6-acetyl-l-lysine myo-inositol, hypotaurine, pantothenic acid (Vitamin B5), lysine, (2e)-oct-2-enediol, butyrylcarnitine, penitricin D. The 14 other metabolites identified (o-acetylleucine, 10-oxodihydrobotry, waraterpol, 5-l-glutamyl-l-leucine, campyrone c, n-acetylleucine, l-norleucine, taurine, isoleucine, 3-(hydroxyacetyl)-indole, citrate and guanine were decrease. In contrast, adding conidia secretome to C. albicans culture induced a switch in the metabolite distribution: the 13 metabolites that were elevated in the culture control and in presence of hyphae secretome became decreased while the 14 metabolites that were decreased with hyphae secretome and in the culture control became increased. Interestingly, virulent linked metabolites: n6-acetyl-l-lysine, a N-acyl-alpha amino acids, pantothenic acid and lysine (metabolite-biofilm related) were decreased in presence of conidia secretome 29 . Although, compounds responsible for remodelling C. albicans cell wall architecture (lactose and lactate) were decreased in presence of conidia secretome. These findings, indicates that conidia secretome may induce changes in C. albicans cell wall architecture (Table 1 ). Also, this conidia-specific metabolic reversal was reflected in the PCA analysis, where conidia secretome-enriched cultures clustered distinctly apart from both control and hyphae secretome conditions. Fungal morphology-specific secretomes induce singular variation in metabolite distribution. Next, we used a volcano plot using (p < 0.05) to highlight individuals features presenting a significantly different change between the monoculture control and the enriched culture with secretomes (Fig. 3 ). In the C. albicans enriched culture with conidia secretome, 26 metabolites were significantly altered (10 metabolites were decreased and 16 metabolites increased) compared to 11 metabolites (7 metabolites decreased and 4 metabolites increased) for the C. albicans enriched culture with hyphae secretome (Fig. 3 a and 3 b). Notably, trehalose, a metabolite produced from mannitol (trehalose biosynthesis pathway: https://pubmed.ncbi.nlm.nih.gov/33511160/#&gid=article-figures&pid=figure-3-uid-2 ) and glutathione, an antioxidant, were in lower concentration in presence of A. fumigatus hyphae secretome in the C. albicans culture (Fig. 3 b). As seen in the heatmap (Fig. 2b), guanine, a nitrogenous base, was increased in presence of conidia and hyphae secretome. Amino acids, such as histidine and lysine were decreased in presence of conidia secretome while isoleucine, beta-alanine and L-norleucine were increased. Speradine B, an alkaloid already isolated from a sponge derived fungus A. flavus MXH-X104 was only present when adding conidia secretome to the C. albicans culture 30 , 31 . A. fumigatus enriched culture supplemented with C. albicans secretome displayed a different metabolites distribution compared to the C. albicans enriched culture (Fig. 3 c and 3 d). Metabolites involved in purine and pyrimidine pathway: inosine monophosphate (IMP), inosine were increased. The vitamin B3 (Niacinamide) was decreased in presence of yeast secretome in A. fumigatus culture. Similarly, IMP, inosine and uridine-5’-monophosphate (UMP) level were increased by hyphae secretome. Interestingly, spirotryprostatin A, an A. fumigatus mycotoxin was decreased in presence of C. albicans hyphae secretome. These results indicate that fungal secretome added to the whole culture ( A. fumigatus or C. albicans ) induce unique metabolomic signature. Conidia secretome seemed to have the most impact on C. albicans culture, since a larger group of metabolites were altered in the enriched culture compared to the other conditions. Carbon markers such as trehalose and glutathione were affected by A. fumigatus hyphae secretome in C. albicans culture. Inhibition of A . fumigatus mycotoxins by C. albicans secretome . The timsTOF LC-MS analysis detected 10 Aspergillus mycotoxins main virulence factors: Sphingofungins B and D, cyclotryprostatins A, spirotryprostatin A, TR-2 belonging to the fumitremorgins family, isomeric quinazoline-containing indole alkaloids such as fumiquinazoline F and D, aculeatsquinone D, alternariol 4-methyl-10-acetyl ether and speradine B (detailed in Table 2 ). To evaluate how mycotoxins were distributed between culture conditions, a metabolic heatmap was generated from timsTOF LC-MS metabolomic data comparing A. fumigatus pure culture with enriched cultures containing hyphae and yeast secretome (Fig. 4 ). Among the 10 mycotoxins identified, three toxins: Spirotryprostatin A, sphingofungin B and D were significantly decreased in presence of C. albicans secretomes. Interestingly, fumiquinazoline F and D, TR-2, aculeatsquinone D, alternariol 4-methyl-10-acetyl ether, speradine B and cyclotryprostatins A levels were comparable in all conditions (culture control and enriched cultures). Thus, molecules present in C. albicans secretome may counteract certain mycotoxins produced by A. fumigatus . Alteration of purine and pyrimidine pathways and cell wall precursors in enriched culture. Enrichment analysis was performed to see which metabolic pathways were the most active in the metabolites produced by the different fungal forms. Among the main class of metabolites specific to A. fumigatus genus, we could identify five significant classes of metabolites: organooxygen compounds, carboxylic acids and derivatives, organonitrogen compounds, purine and pyrimidine pathway. Significant metabolites belonging to purine and pyrimidine pathways were produced in both A. fumigatus and C. albicans enriched cultures (Fig. 5 a, d and 6 a, e). These specific pathways are essential in both species for virulence and growth 32 – 36 (Table 3 ). In the purine pathway, the presence of yeast secretome in A. fumigatus culture showed a variation of metabolites concentration belonging to the salvage pathway: IMP ( p = 0.0025) and inosine ( p = 0.0116) were significantly increased whereas adenosine monophosphate (AMP) ( de novo purine pathway) was decreased (Fig. 5 b). Hyphae secretome showed the same variation of metabolites in A. fumigatus culture: significant increase of inosine ( p = 0.0175) and IMP ( p = 0.0104). The other metabolites stayed at the same level (Fig. 5 e). The purine pathway biosynthesis steps for fungal pathogens are visible here: https://pmc.ncbi.nlm.nih.gov/articles/PMC5488104/figure/microorganisms-05-00033-f001/ . In the pyrimidine pathway (for A. fumigatus : https://www.researchgate.net/publication/340602879/figure/fig1/AS:11431281432025133@1746830936226/The-de-novo-pyrimidine-biosynthesis-pathway-which-leads-to-the-formation-of-UMP-In.tif ), both yeast and hyphae secretome added to A. fumigatus culture similarly increased UMP ( p = 0.0318) and decreased uridine diphosphate-N-acetylglucosamine (UDP-N-acetylglucosamine) (precursor of chitin, a component of fungi cell wall). The other metabolites linked to pyrimidine pathway stayed at the same level: uridine diphosphate glucose (UDP-glucose, precursor of β-glucans, cell wall component) and uridine diphosphate-galactose (UDP-galactose) (Fig. 5 c and 5 f). A. fumigatus secretome added to C. albicans culture showed a different variation of metabolites from purine pathway compared to C albicans enriched culture with A. fumigatus secretome. ADP (salvage pathway) ( p = 0.00248) and 2’-O-methyladenosine ( p = 0.00297) were significantly increased while AMP ( de novo purine pathway) and inosine (salvage pathway) were decreased by conidia secretome (Fig. 6 b). Hyphae secretome showed the same increased of compounds as with conidia secretome belonging to the salvage pathway: ADP, adenosine ( p = 0.0171) and IMP. 2’-O-methyladenosine while AMP and inosine abundance did not change (Fig. 6 f). In pyrimidine pathway, both conidia and hyphae secretome decreased both A. fumigatus conidia and hyphae secretome decreased UDP-N-acetylglucosamine as observed in A. fumigatus enriched culture. UDP-galactose was significantly increase in C. albicans enriched culture with hyphae secretome. UMP and UDP-glucose abundance was unchanged compared to the culture control (Fig. 6 c, g). Steps of C. albicans pyrimidine pathway are visible here: https://www.kegg.jp/pathway/cal00240 . A. fumigatus and C. albicans showed different variation of metabolites belonging to purine and pyrimidine pathway that could be caused by stress. Cell wall components, especially UDP-N-acetylglucosamine was affected by all fungal secretome in enriched cultures. Selective induction of indole pathway metabolites in C. albicans culture enriched with A. fumigatus secretome . Key indole metabolites were significantly identified in C. albicans enriched culture but were absent in enriched A. fumigatus culture: tryptophol, indoleacetaldehyde, indoleacrylic acid, 5-hydroxytryptophol, indole-3-carboxaldehyde, 5-hydroxy-L-tryptophan and L-tryptophan (Fig. 6 d, h). Indoles metabolites in C. albicans may be involved in the synthesis of quorum sensing molecule, here, tryptophol, to regulate the population in response to stress (insufficient nutrients available). Tryptophol level was decreased in presence of conidia secretome (Fig. 6 d). Overall, the metabolites associated with indoles derivatives pathway were significantly decreased in presence of conidia secretome significantly decreased L-tryptophan ( p = 0.00249), indoleacrylic acid ( p = 0.0149) and 5-hydroxy-L-tryptophan ( p = 0.00195), while indole-3-carboxaldehyde was significantly increased ( p = 0.0294). Hyphae secretome decreased as well significantly indoleacetaldehyde ( p = 0.00613) and 5-Hydroxy-L-tryptophan but the other metabolites stayed at the same level (Fig. 6 h). The presence of indole metabolites in C. albicans enriched culture is specific to the production of quorum sensing molecule (tryptophol) in response to the presence of A. fumigatus secretome. Stress resistance is modulated by carbon adaption. Organooxygen metabolites essential for fungal energetic metabolism were expressed in A. fumigatus and C. albicans enriched cultures ( Supplementary Fig. 2 ). In A. fumigatus enriched culture, the level of glycerol in presence of yeast secretome was significantly increased ( p = 0.0121) while D-glucose level was unchanged. In presence of hyphae secretome, glycerol level was unchanged, and D-glucose was slightly increased. There was no trehalose detected whether yeast or hyphae secretome was added ( Supplementary Fig. 2a). The distribution of metabolites belonging to organooxygen pathway for the C. albicans culture supplemented with A. fumigatus secretome was different ( Supplementary Fig. 2b ). Organooxygen metabolites plays an important role in C. albicans cell wall architecture. Glycerol level was decreased in presence of conidia secretome while D-glucose and trehalose were increased. Enriched culture with hyphae secretome showed different variation of these metabolites: there was a significant decrease of trehalose ( p = 0.0112). D-glucose was slightly increased while glycerol was decreased. Variation of these carbon-linked metabolites may have induced structural modifications in C. albicans cell wall upon exposure to A. fumigatus secretomes. These variations in cell wall structure could impact fungal response to stress. DISCUSSION The nature of polymicrobial interaction —whether competitive, antagonistic, or mutualistic— critically influences disease severity and patient outcome. Our study provides the first multimodal metabolomics-based characterization of the interaction between A. fumigatus and C. albicans revealing antagonistic behaviour and raising the possibility of mutualistic coexistence. This dual interaction paradigm has direct clinical implications for management of immunocompromised patients, where both pathogens may co-occur. We chose to focus on secretomes in our enriched cultures experiment to specifically capture the metabolites released by distinct fungal morphological forms. This approach directly models the clinical scenario: during infection, the host is simultaneously exposed to multiple fungal morphotypes —yeast, conidia, and hyphae— which coexist and release different repertoires of metabolites 37 , 38 . Understanding these morphotype-specific contributions is essential for predicting disease progression, as each form may differentially modulate the coculture environment and host response. C. albicans culture supplemented with conidia secretome formed a distinct metabolite cluster reflecting a dormant state, characterized by low respiratory activity, reduced amino acid levels, and trehalose storage—typically metabolized during germination 39 . By contrast, C. albicans culture enriched with hyphal secretome displayed metabolic activation with increased lactate and amino acid biosynthesis, consistent with the energy demands of rapid growth and secondary metabolite production 39 . Metabolite levels linked to pyrimidine and purine pathway varied under enriched cultures compared with the pure culture for all conditions suggesting metabolic stress. This represents the first layer of antagonism: competition for shared resources in the medium. The metabolite profiles could have indicated a shift toward de novo biosynthesis of purines and pyrimidines—a less energy-efficient strategy typically triggered by nutrient limitation. Specifically, UMP ( de novo pyrimidine pathway) was increased while UDP-N-acetylglucosamine, essential for cell wall construction in both fungi, was decreased 32 . In the metabolites belonging to the de novo purine pathway: AMP level was reduced and ADP and IMP increased, reflecting altered energy status and metabolic flux through this biosynthetic route. Together, these changes suggest that resource competition forces both fungi to rely on metabolically costly de novo synthesis pathways for survival 32 , 33 , 40 , 41 . Beyond metabolic competition, a second layer of antagonism emerged: a direct chemical warfare through secreted antifungal compounds. On solid media, A. fumigatus growth was restricted by C. albicans , producing a “distance-inhibition” zone with reduced hyphal density 42 – 45 . This pattern, consistent with prior observations of inhibitory activity 28 , 46 , suggests active release of antifungal molecules, which our metabolomics approach confirmed through identification of tryptophol. Tryptophol is a quorum-sensing molecule produced by C. albicans that represses hyphal development and inhibits both fungi 47 – 50 . The growth limitation of A. fumigatus thus results from a dual mechanism: (i) metabolic stress from nutrient competition forcing switch to energy-expensive de novo biosynthesis, and (ii) direct chemical inhibition through tryptophol release. Conversely, A. fumigatus secreted mycotoxins with antifungal activity against C. albicans , especially sphingofungins B, Fumiquinalozine D and spirotryprostatin A. Sphingofungins disrupt sphingolipids biosynthesis in the fungal cell wall 5 , 51 – 53 , while spirotryprostatin A exerts antimitotic and antimicrobial activity 31 , 54 , 55 . Interestingly, both mycotoxins (spirotryprostatin A and sphingofungin B) were reduced in enriched culture with C. albicans secretome (yeast and hyphae), suggesting possible metabolic counteractions by C. albicans. A critical clinical concern emerged from our data: antagonistic fungal interactions generated metabolites that may directly worsen patient outcomes. We identified indole-derived metabolites, particularly indoleacrylic acid, indole-3-acetaldehyde, and indole-3-carboxyaldehyde released in C. albicans culture supplemented with A. fumigatus secretome. These metabolites may be part of tryptophan-independent pathway, which exist in fungi species and may be expressed in yeast strains 56 . Recent evidence linked these metabolites to severe clinical outcomes in pneumonia 57 . Specifically, indole-3-acetaldehyde (which converts to indole-3-acetic acid), and its derivative indole-3-acetic acid have been associated with increased pneumonia severity (higher pulmonary damages, production of reactive oxygen species, increase of immune resistance), higher hospitalization risk, and elevated mortality rates 1 , 57 . Given that ventilator-associated pneumonia and bloodstream infections are the predominant intensive care unit (ICU)-acquired infections in immunocompromised patients mainly caused by A. fumigatus and C. albicans and the production of indole metabolites during fungal interactions may significantly exacerbate disease severity and contribute to poor clinical outcomes 1 . Although antagonistic competition dominates the metabolic landscape, our data revealed mechanisms by which these pathogens may simultaneously support each other. The detected A. fumigatus mycotoxins fumiquinalozines A and cycloprostatin A are known to be associated with biofilm formation 5 . Their unchanged levels in enriched culture with the C. albicans secretome could suggests that A. fumigatus can maintain biofilm formation in the presence of the yeast. DL-pipecolate, a metabolite involved in C. albicans biofilm remained also unchanged in presence of A. fumigatus secretome 58 . The previously indole metabolite involved in pneumonia exacerbation: indole-3-acetaldehyde which converts to indole-3-acetic acid, is also known to promote fungi filamentation and biofilm 56 . Despite A. fumigatus and C. albicans having distinct biofilms 59 , both fungi produce biofilms rich in lipids and extracellular DNA and share the same chitin precursor (UDP-N-acetylglucosamine) and β-glucans precursor (UDP-glucose) 60 , which may enable structural interconnection, providing a potential mechanism by which C. albicans stabilizes or enhances A. fumigatus biofilm formation 21 . Mixed biofilms are common in polymicrobial infections and contribute to pathogens persistence through combined protective matrix and antifungal resistance 2 , 21 , 61 . Some mycotoxins identified in this study are established tissue-damaging agents that compromise pulmonary tissue integrity 52 , 62 , 63 creating a disrupted host niche conducive to enhanced colonization and modify disease progression. Together, mycotoxin-induced tissue damage and mixed biofilm-mediated persistence enable synergistic interactions that may significantly exacerbate infection severity 13 . While our study highlighted antagonistic behaviour between A. fumigatus and C. albicans , co-infections pathogens usually interact synergistically and support each other 2 , 13 , 64 . Since C. albicans and A. fumigatus tolerated each other in the same environment (in solid medium), mutualism between the two species could also exist despite their antagonistic behaviour. To examine this possibility, we recorded a 12 h liquid coculture in a slide chamber and confirmed that both fungi remained viable. The C. albicans inoculum increased considerably in the presence of A. fumigatus , and both species formed hyphae and pseudohyphae. We also observed the formation of multicellular clusters from C. albicans in the presence of A. fumigatus , a virulence-associated stress response 38 ( Supplementary Fig. 3 ). This experimental evidence is supported by clinical observations: Kubota et al. , (2023) reported invasive pulmonary aspergillosis combined with candidiasis in an immunocompromised patient, where yeasts were embedded within A. fumigatus hyphal networks 4 . The embedding of yeast cells within the hyphae suggests a mutualistic interaction, highlighting that these two fungi can coexist and adapt to one another in the same niche. Although this work significantly advances understanding of A. fumigatus - C. albicans interactions, it is not without limitations. The metabolomic measurements of secretomes added to enriched cultures were excluded because the metabolite profiles were inconsistent or of poor quality. We employed three different culture media with distinct purposes: AMM and YPD to maximize metabolite production in enriched cultures, and PDA as a neutral basal medium for fungal coculture. Metabolite profiles in some secretome conditions showed minimal differences from monocultures, likely due to lower secretome concentration. CONCLUSION Using a novel multimodal metabolomics approach, this study demonstrates that A. fumigatus and C. albicans engage in complex secretory metabolic interactions characterized by both potential antagonism and mutualism. For immunocompromised populations, where these fungi represent the predominant invasive pathogens, their capacity for coexistence presents a critical clinical threat. Colonization by one species creates a permissive niche for the other; compounding this risk, the production of tissue-damaging mycotoxins, inflammatory indole metabolites that exacerbate pneumonia, and mixed biofilms collectively drive severe, treatment-resistant infections. The vulnerability of immunocompromised patients is further heightened by the clinically indistinguishable symptoms of mono- and polymicrobial infections, which impede accurate diagnosis and delay appropriate dual-species therapy, ultimately worsening prognosis and increasing mortality. Understanding these metabolic mechanisms is essential for developing targeted therapeutic strategies that disrupt fungal interactions and improve outcomes in vulnerable populations. Future studies in clinical settings mimicking coinfection are needed to elucidate mutualism mechanisms, particularly the role of mixed biofilm formation and dosage-dependent functional impact of metabolites. METHOD details A. fumigatus and C. albicans strains and growth conditions. A. fumigatus wild-type strain ATCC 46645 and C. albicans SC5314 (kindly sent by the University Bourgogne Franche-Comte in Dijon) were used in this work. For the culture control, A. fumigatus was grown on Aspergillus minimal media (AMM) and C. albicans on yeast peptone dextrose broth (YPD) (Sigma-Aldrich, Damstadt, Germany) (500 \(\mu\)L of 10 6 cells/mL spread on Petri dish for both fungi). Enriched cultures were grown in the same conditions supplemented with secretome for the 1 H-NMR and TimsTOF LC-MS analysis. Coculture containing both whole strains were grown on Potato dextrose agar extract (PDA) (Sigma-Aldrich, Damstadt, Germany) 10 days at 25°C to allow complete growth of both fungi and have fungal morphological pattern. A. fumigatus secretomes. Conidia suspensions were obtained by harvesting grown mycelia on AMM plates with phosphate buffer saline PBS-T (DPBS, Gibco Life technologies, Carlsbad, CA, USA) (Tween 20 0.01%) followed by filtration through 100 µm and 30 µm filters (Miltenyi Biotec, Bergisch Gladbach, Germany). For conidia secretome preparation, 10⁶ conidia/mL were incubated for 6–8 h at 30°C. Cultures were then centrifuged centrifuged at 3,000 × g for 10 min at room temperature (RT), and supernatants were collected and filtered (100 µm and 30 µm) to remove residual conidia. For A. fumigatus hyphal secretome, 10⁶ conidia/mL were inoculated in RPMI medium and incubated at 37°C for 16–18 h to allow complete hyphal development. Supernatants were collected by centrifugation at centrifuged at 3,000 × g for 10 min at RT and filtered as described above. C. albicans secretomes. For yeast secretome, yeasts were collected from an overnight culture, washed twice with YPD (4,600 × g for 3 min at RT), and adjusted to 10⁶ cells/mL (OD₆₀₀). Yeast suspension was incubated during 6h-8h at 30°C to generate metabolites. For hyphal secretome, yeast cells at 10⁶ cells/mL were incubated at 37°C for 16–18 h in RPMI to induce hyphal development. Both secretomes were then centrifuged at 3,000 × g for 10 min at RT, and supernatants were collected and sequentially filtered (100 µm and 30 µm) to remove residual cells. To ensure secretome purity, cultures were microscopically examined before centrifugation to confirm the presence of a single morphological form (100% yeast, conidia, or hyphae). Only cultures displaying 100% morphotype homogeneity were retained for secretome collection. Following centrifugation, supernatants were additionally filtered if any residual cells were detected. Representative microscopy images confirming secretome purity and morphotype specificity are provided in (Fig. 7). Enriched cultures of A. fumigatus and C. albicans . To assess the impact of secretomes on fungal metabolism, enriched cultures were prepared by adding secretomes to pre-established cultures. For enriched A. fumigatus cultures, 10⁶ conidia/mL were pre-grown on Petri dishes for 1 day at 37°C to allow initial growth, then 800 µL of C. albicans secretome (yeast or hyphal) was added. For enriched C. albicans cultures, 10⁶ yeast cells/mL were pre-grown for 1 day at 30°C, then 800 µL of A. fumigatus secretome (conidial or hyphal) was added. All plates (monocultures and enriched cultures) were subsequently incubated for 5 days at 25°C. A. fumigatus and C. albicans coculture . To observe fungal morphological pattern, solid coculture using both fungi was performed. 100 µL of each species was point inoculated on the extremity of the Petri dish at 1x10 6 cells/mL. The cultures were incubated 10 days at 25°C. Samples collection procedure. The enriched cultures and monocultures were rinse two times with 5 mL pre-cooled (4°C) PBS. PBS was removed quickly, and samples collected into 15 ml falcon tubes and pour quickly in liquid nitrogen. Samples were then directly transferred to ice. 0.8 mL of pre-chilled at -80°C methanol was added to the falcon tubes. The samples were vortex strongly for 10 sec and placed on dry ice. All sample were stored at -80°C until measurements. Only the cell pellets were used for multimodal metabolomics analysis. Metabolites extraction and 1 H-NMR analysis . The extracts were transferred into 2 mL AFA™ Covaris glass tubes. Each sample was thoroughly mixed by vortexing with 900 µL methanol, 100 µL chloroform, and 100 µL ultrapure water before being loaded in the Covaris ultrasonicator E220 Evolution. Following extraction, solutions were loaded into a centrifuge (12,000g force, 30 min at 6°C) for complete phase separation. Centrifuged samples were later manually collected with a mechanical pipette. The chloroform layer was transferred to an HPLC vial, while the polar phase was placed into an Eppendorf tube. Polar phase samples were dried overnight under vacuum concentrator (SpeedVac: Preset 2 until complete solvent evaporation was achieved. Dried pellets of polar phase solutions were resuspended in 50 µL of deuterated phosphate buffer (200 mM K 2 HPO 4 , 200 µM NaN 3 , pH 7.4) with 1 mM internal standard TSP. The reconstituted solutions were sonicated for 1 min in Eppendorf tubes, then centrifuged at 30,000 × g for 30 min to eliminate residual particulate matter. Aliquots of 40 µL from the supernatant were transferred into 5 mm NMR tubes using gel loading pipette tips and placed in a sample rack. The samples were stored at 4°C until NMR spectral acquisition. Proton nuclear magnetic resonance (¹H-NMR) spectra were reacquired by a 14.10 Tesla (600 MHz for proton channel) ultra-shielded NMR spectrometer with a 5 mm triple resonance TXI room temperature probe (Avance™ III HD, Bruker BioSpin, Karlsruhe, Germany). For metabolomics analysis, Carr-Purcell-Meiboom-Gill (CPMG) was employed with 256 scans and water suppression used for spectral measurements at 298 K (24.85°C). The recorded free induction decays (FIDs) were Fourier-transformed (FT); phase and baseline corrections were applied. Metabolites annotation and quantification was performed employing commercial software (ChenomX NMR Suite 9.0) and integrated databases (ChenomX and HMDB libraries). Statistical analysis of metabolomics data was performed using MetaboAnalyst 5.0 R-based online analysis tool (http://www.metaboanalyst.ca/). Metabolites were excluded from the analysis if more than 66% of the results for a metabolite were missing. For the remaining metabolites, missing values were replaced with a small value corresponding to 1/5 of the minimum positive value of that metabolite in the original dataset. To account for dilution effects, the data were normalized to a reference sample using probabilistic quotient normalization (PQN) and scaled using Pareto scaling (mean-centred and divided by the square root of its standard deviation of each variable). TimsTOF LC-MS measurements. For comprehensive LC-MS analysis, a chromatography system (Elute Plus, Bruker Daltonics, Bremen, Germany) was coupled to a trapped ion mobility spectrometry time of flight (timsTOF) mass spectrometer (timsTOF Pro 2, Bruker Daltonics, Bremen, Germany) equipped with a vacuum insulated probe heated electrospray ionization (VIP-HESI) source. The instruments were controlled using the Bruker HyStar software and TimsControl acquisition software. Reversed-phase (RP) chromatography was used for the analysis employing an Intensity Solo 2 C18 column (100 mm × 2.1 mm, 2 µm; Bruker Daltonics) was used. The eluent system for mobile phase A was water with 0.1% formic acid, and for mobile phase B, ACN with 0.1% formic acid. The flow rate was set to 0.6 mL/min with a gradient starting at 5% B for the first 2 min, increasing to 60% B at 10 min, 98% B at 11 to 13 min, and returning to 5% B at 13.1 minutes until 15.5 min. The injection volume was 2 µL of the desalted samples. The MS analysis for non-targeted metabolomics was performed using Parallel Accumulation Serial Fragmentation (PASEF) mode with data-dependent MS/MS acquisition and Trapped Ion Mobility Spectrometry (TIMS) stepping, following the 4D metabolomics standard method in TimsControl software (Bruker Daltonics, Bremen, Germany). The source parameters were set as follows: End Plate Offset at 500 V, Capillary Voltage at 4500 V, Nebulizer Pressure at 2 bar, Dry Gas Temperature at 230°C, Dry Gas Flow at 8.0 L/min, Sheath Gas Temperature at 400°C, and Sheath Gas Flow at 4 L/min. The specific timsTOF Pro 2 parameters included the following: Acquisition Mode in PASEF with TIMS on, Number of PASEF Ramps set to 2, Mass Range from 50–1300 Da, Mobility Range (1/K₀) from 0.10–1.50 V·s/cm², TIMS Ramp Time at 100 ms, Collision RF at 450 Vpp, TOF Transfer Time at 65 µs, Pre Pulse Storage Time at 3 µs, and TIMS Stepping enabled with two steps. The collision energy for fragmentation was set to 20/50 eV. RP chromatography was conducted in both negative and positive ionization mode. Mass and mobility were recalibrated using a 1:3 (v/v) mixture of sodium formate and Agilent’s ESI-L LC/MS Tuning Solution, injected at the start of each run. Additionally, a pooled QC sample (containing same amount from each sample) was used for signal correction during MS analysis. MS data processing and metabolite annotation were performed using MetaboScape 2025 software (Bruker Daltonics, Bremen, Germany). Features have been extracted utilizing the program’s T-ReX-4D™ algorithm with an intensity threshold of 3000 ion counts and minimum 4D peak size of 150 points. Recursive feature extraction was activated with a minimum 4D peak size of 125 points. Metabolites were assigned by matching extracted features against target lists (Bruker HMDB Metabolite Library 2.0, METLIN-CCS Lipid Database, PNNL CCS Metabolites Database, Unified CCS Compendium 2020-03-30, Microbial Metabolites Database Version 1.0) and spectral libraries (Bruker MetaboBASE Personal Library 3.0). The annotations followed MS/MS-based level 2 confidence criteria for metabolite identification, as defined by the Metabolomics Standards Initiative 65 . A match was considered if, in addition to the mass-to-charge ratio (m/z) deviation being < 2.0 ppm, at least one of the following criteria was met: mSigma < 20, MS/MS score < 900, or collision cross-section (CCS) deviation < 1%. The extracted ion chromatograms shapes were also considered. Features with intensities in study samples less than three times those observed in blanks were excluded. Each measurement mode was pre-processed using the MetaboAnalyst web server (Version 6.0, www.metaboanalyst.ca ). Data were uploaded as comma-separated value (.csv) files, with missing values being replaced with 1/5 of the minimum positive value for each feature. Raw data was log transformed to stabilize variance and reduce skewness for each RP mode separately. Features detected by multiple methods were retained from the method with the lowest number of missing values and the highest overall signal intensity. Table 1 Summary of C. albicans metabolites associated with virulence and pathogenicity detected in C. albicans enriched culture with A. fumigatus secretome. Metabolites are described with their class, metabolite ID (from https://mimedb.org) and their biological function. Metabolite Class Metabolite ID Biological function DL-Pipecolate metabolite of lysine MMDBc0000492 Biofilm formation 58 N6-Acetyl-L-lysine N-acyl-alpha amino acids MMDBc0000503 Identified in Candida maltose . The enzyme was strongly induced in cells grown on L-lysine as the sole carbon source 29 Pyridoxamine the 4-aminomethyl form of vitamin B6 MMDBc0000195 Virulence in C. albicans and A. fumigatus 66,67 D-pantothenic acid Vitamine B5 MMDBc0000069 Essential for C. albicans virulence and yeast survival ( A. fumigatus : virulence and siderophore production) 66,67 Niacinamide Vitamine B3 MMDBc0000192 Component of the coenzyme NAD in vitamin biosynthesis pathway 66,67 Piridoxine The 4-methanol form of vitamin B6 MMDBc0000081 Virulence in C. albicans and A. fumigatus 66,67 Tryptophol indolyl alcohol MMDBc0007020 Fungal quorum sensing molecule 48,49 Trehalose 1-alpha (disaccharide) sugar MMDBc0000177 Role in resistance to environmental stress, virulence and survival 40,68,69 L-acetylcarnitine Acyl carnitines MMDBc0000501 Role in invasion: Affect claudin subtypes (open intestinal tight junctions) 70 L-phenylalanine L-alpha-amino acid MMDBc0000048 Uptake in differenciated hyphae 71 Lysine Amino acid MMDBc0000059 Biofilm related pathway 72 Glutathione antioxidant MMDBc0029474 Modulation stress resistance 73 Lactate secondary alcohols MMDBc0056071 Cell wall remodelling: thicker cell wall in C. albicans and reduce immune cell visibility 73,74 Table 2 Table summarizing all the detected Aspergillus mycotoxins by TOF tims LC-MS with their class, metabolite ID (from https:/mimedb.org) and their biological function. Metabolite Class Metabolite ID Biological function Spirotryprostatin A Diketopiperazine alkaloids MMDBc0001046 Cytotoxicity, antimicrobial effects, antifungal properties 54,55,75,76 Sphingofungin B Long-chain fatty acids MMDBc0019721 Similar role as fumonisins, antifungal activity 53,77 Sphingofungin D Phytoceramides MMDBc0002934 Antifungal activity, inhnibit enzyme essential in the biosynthesis of sphingolipids 53 Cyclotryprostatin A Beta carbolines MMDBc0013469 Inhibitor of the mammalian cell cycle 76,78 TR-2 verruculogen Beta carbolines, fumitremorgins family MMDBc0012435 Tremorgenic mycotoxin 79,80 Fumiquinazoline F Peptidyl alkaloid MMDBc0019494 Cytotoxic effect, produced during biofilm formation 81–83 Fumiquinalozine D Quinalozine CID 9980845 Antibacterial and antifungal ( C. albicans ) 82–84 Aculeatsquinone D Benzoquinone derivatives MMDBc0009940 Cytotoxic, antimicrobial, antifungal 85,86 Alternariol 4-methyl-10-acetyl ether Phenolic compound MMDBc0006882 Cytotoxic 87 Speradine B Cyclopiazonic acid alkaloids MMDBc0001931 Cytotoxic activity 30,31 1-pyrroline-4-hydroxy-2-carboxylate Pyrrolinecarboxylic acid MMDBc0049981 Heat tolerance in fungi 88 6.7-seco-Agroclavine Alkaloid clavines and derivatives MMDBc0013361 Unknown 89 Table 3 Significant metabolites found in essential fungal pathway (here in C. albicans and A. fumigatus ) with their class, metabolite ID, biological function and metabolic pathway (from https://mimedb.org). Metabolite Class Metabolite ID Biological function Metabolic Pathway Inosine Purine mucleoside MMDBc0000064 Second messenger 41 Salvage pathway Guanine Derivative of purine MMDBc0000037 Hydrolyze bound GTP to GDP 41 Salvage pathway UMP Uracil nucleotide MMDBc0029491 Precursor for RNA synthesis 33 Pyrimidine pathway Uridine Pyrimidine nucleoside MMDBc0000094 Convert to UMP, crucial for virulence and survival in the host 33 Pyrimidine pathway UDP-glucose Pyrimidine nucleotide sugars MMDBc0032979 Beta-glucans precursor cell wall (C. albicans and A. fumigatus) 32 Pyrimidine pathway UDP-N-Acetylglucosamine Nucleotide sugar MMDBc0029492 Chitin precursor cell wall ( C. albicans and A. fumigatus) 32 Pyrimidine pathway Indoleacetaldehyde 3-alkylindoles MMDBc0032968 Derived tryptophan, Convert to Indole-3-acetic acid 41,90,91 Indole and derivatives pathway Indole-3-carboxaldehyde Indole MMDBc0000271 Derived tryptophan, Convert to indole-3-carboxylic acid 41 Indole and derivatives pathway (S)-8-amino-7-oxononanoic acid Medium-chain fatty acids MMDBc0054107 Substrate in biotin pathway https://pubchem.ncbi.nlm.nih.gov/compound/7-Keto-8-Aminopelargonic-Acid) Biotin pathway. Declarations Author contributions Conceptualization was done by NB, CT, FD, ST. Data Curation completed by ST, DI and JS. Formal Analysis was done by ST and GB. ST performed Investigation. CT did the research Validation. Supervision was under FD, NB and CT. Writing-Original Draft Preparation was done by ST. Writing-review and Editing was done by NB, CT and FD. Acknowledgements We thank Daniele Bucci for their technical support as well as Sisi Deng. Illustrations were in part generated using Biorender. This study was funded by the Deutsche Forschungsgemeinschaft (German Research Foundation Project 512332979), Bruker Switzerland AG (grant number B23F-1D9C) and the Werner Siemens Foundation. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Declaration of interests CT reports a research grant by Bruker Switzerland AG. 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LC-MS analysis reveals biological and metabolic processes essential for Candida albicans biofilm growth. Microb. Pathog. 152, 104614 (2021). Brown, A. J. P., Brown, G. D., Netea, M. G. & Gow, N. A. R. Metabolism impacts upon Candida immunogenicity and pathogenicity at multiple levels. Trends Microbiol. 22, 614–622 (2014). Ballou, E. R. et al. Lactate signalling regulates fungal β-glucan masking and immune evasion. Nat. Microbiol. 2, 16238 (2016). Rateb, M. E. et al. Induction of diverse secondary metabolites in Aspergillus fumigatus by microbial co-culture. RSC Adv. 3, 14444 (2013). Maiya, S., Grundmann, A., Li, S. & Turner, G. The Fumitremorgin Gene Cluster of Aspergillus fumigatus : Identification of a Gene Encoding Brevianamide F Synthetase. ChemBioChem 7, 1062–1069 (2006). Vanmiddlesworth, F. et al. Sphingofungins A,B,C, and d; a new family of antifungal agents. I. Fermentation, isolation, and biological activity. J. Antibiot. (Tokyo) 45, 861–867 (1992). Cui, C.-B., Kakeya, H. & Osada, H. Novel Mammalian Cell Cycle Inhibitors, Cyclotryprostatins A-D, Produced by Aspergillus fumigatus, Which Inhibit Mammalian Cell Cycle at G2/M Phase. Li, X.-J., Zhang, Q., Zhang, A.-L. & Gao, J.-M. Metabolites from Aspergillus fumigatus , an Endophytic Fungus Associated with Melia azedarach , and Their Antifungal, Antifeedant, and Toxic Activities. J. Agric. Food Chem. 60, 3424–3431 (2012). Bott, A. et al. In vitro transformation of the tremorgenic mycotoxin verruculogen. Mycotoxin Res. 8, 2–8 (1992). Lim, F. Y., Ames, B., Walsh, C. T. & Keller, N. P. Co-ordination between BrlA regulation and secretion of the oxidoreductase FmqD directs selective accumulation of fumiquinazoline C to conidial tissues in A spergillus fumigatus : Fumiquinazoline biosynthesis in Aspergillus fumigatus . Cell. Microbiol. 16, 1267–1283 (2014). Yurchenko, A. N. et al. The Metabolite Profiling of Aspergillus fumigatus KMM4631 and Its Co-Cultures with Other Marine Fungi. Metabolites 13, 1138 (2023). He, F. et al. Indole alkaloids from marine-derived fungus Aspergillus sydowii SCSIO 00305. J. Antibiot. (Tokyo) 65, 109–111 (2012). Afiyatullov, Sh. Sh., Kalinovskii, A. I., Pivkin, M. V., Dmitrenok, P. S. & Kuznetsova, T. A. Alkaloids from the Marine Isolate of the Fungus Aspergillus fumigatus. Chem. Nat. Compd. 41, 236–238 (2005). Chen, L. et al. Aculeatusquinones A-D, Novel Metabolites from the Marine-Derived Fungus Aspergillus aculeatus. HETEROCYCLES 87, 861 (2013). Evidente, A. Advances on anticancer fungal metabolites: sources, chemical and biological activities in the last decade (2012–2023). Nat. Prod. Bioprospecting 14, 31 (2024). Den Hollander, D. et al. Cytotoxic Effects of Alternariol, Alternariol Monomethyl-Ether, and Tenuazonic Acid and Their Relevant Combined Mixtures on Human Enterocytes and Hepatocytes. Front. Microbiol. 13, 849243 (2022). Xu, K., Yuan, X.-L., Li, C. & Li, A. X.-D. Recent Discovery of Heterocyclic Alkaloids from Marine-Derived Aspergillus Species. Mar. Drugs 18, 54 (2020). Gerhards, N., Neubauer, L., Tudzynski, P. & Li, S.-M. Biosynthetic Pathways of Ergot Alkaloids. Toxins 6, 3281–3295 (2014). Sugiyama, Y., Ito, Y., Suzuki, M. & Hirota, A. Indole Derivatives from a Marine Sponge-Derived Yeast as DPPH Radical Scavengers. J. Nat. Prod. 72, 2069–2071 (2009). Tang, J. et al. Biosynthetic Pathways and Functions of Indole-3-Acetic Acid in Microorganisms. Microorganisms 11, 2077 (2023). Additional Declarations Competing interest reported. C.T. reports a research grant by Bruker Switzerland AG. Supplementary Files Supplementaryinformation.pdf image1.png Graphical Abstract (UDP-GlcNAc: Uridine diphosphate-N-acetylglucosamine and UDP-glucose: uridine diphosphate glucose). Scheme created with Biorender. 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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-9059703","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":626158322,"identity":"94cbe22e-c7c1-4992-81a7-e33d1dbf3bbe","order_by":0,"name":"Sophie Tonneau","email":"","orcid":"","institution":"University Hospital Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Tonneau","suffix":""},{"id":626158323,"identity":"320a610b-449d-4968-8da6-594951fb2f8b","order_by":1,"name":"Denis Ispan","email":"","orcid":"","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Denis","middleName":"","lastName":"Ispan","suffix":""},{"id":626158324,"identity":"6d44baec-14d2-4aaa-9653-e88215b39afa","order_by":2,"name":"Gyuntae Bae","email":"","orcid":"","institution":"University Hospital Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Gyuntae","middleName":"","lastName":"Bae","suffix":""},{"id":626158325,"identity":"fa1a78f9-d6d6-493a-b351-e80675f0d1a5","order_by":3,"name":"Jannik Sprengel","email":"","orcid":"","institution":"University of Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Jannik","middleName":"","lastName":"Sprengel","suffix":""},{"id":626158326,"identity":"6a8b35a6-0894-456f-9b0f-9ca6ed7559d0","order_by":4,"name":"Frederic Dalle","email":"","orcid":"","institution":"Université Bourgogne Franche-Comté","correspondingAuthor":false,"prefix":"","firstName":"Frederic","middleName":"","lastName":"Dalle","suffix":""},{"id":626158327,"identity":"cffae77d-c253-4847-95d0-f15f0066a4dd","order_by":5,"name":"Nicolas Beziere","email":"","orcid":"","institution":"University Hospital Tübingen","correspondingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Beziere","suffix":""},{"id":626158328,"identity":"52ac2f32-b64b-43b1-86b2-de0c50837948","order_by":6,"name":"Christoph Trautwein","email":"data:image/png;base64,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","orcid":"","institution":"University Hospital Tübingen","correspondingAuthor":true,"prefix":"","firstName":"Christoph","middleName":"","lastName":"Trautwein","suffix":""}],"badges":[],"createdAt":"2026-03-07 16:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9059703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9059703/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107452863,"identity":"d229d0e0-aef4-4b99-aaf7-b20038e9581d","added_by":"auto","created_at":"2026-04-21 15:30:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":665042,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental workflow and inhibition fungal pattern obtained after ten days coculturing.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Schematic workflow of the enriched culture set up\u003cstrong\u003e \u003c/strong\u003eperformed with \u003cem\u003eC. albicans \u003c/em\u003esecretome (yeast or hyphae) added to the \u003cem\u003eA. fumigatus\u003c/em\u003e culture. The same method was used with \u003cem\u003eA. fumigatus\u003c/em\u003e secretome (conidia or hyphae) added to \u003cem\u003eC. albicans\u003c/em\u003e culture. Enriched cultures were incubated 5 days at 25 °C to allow generation of metabolites and then \u003csup\u003e1\u003c/sup\u003eH-NMR and timsTOF LC-MS analysis were performed on the supernatants after extraction. In coculture, both whole fungi were added and incubated at 25 °C for 10 days to allow complete grow in the agar plate. \u003cstrong\u003eb\u003c/strong\u003e\u003cem\u003e \u003c/em\u003eGrowth morphologies and colony-front pattern of \u003cem\u003eA. fumigatus \u003c/em\u003eand \u003cem\u003eC. albicans \u003c/em\u003ein coculture during 10 days at 25 °C. Scale bar: 3.44 cm. The ten pictures represent each day of incubation from left to right and top to bottom. Illustrations generated with Biorender.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/57719a253650b1542d9daf3c.png"},{"id":107452865,"identity":"7c7b056a-87e1-49fb-904e-0876caf901b0","added_by":"auto","created_at":"2026-04-21 15:30:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e conidia secretome leads to shift in metabolites distribution. a\u003c/strong\u003e Discriminant analysis score plot of metabolites obtained from pure culture \u003cem\u003eA. fumigatus \u003c/em\u003e(red circles) and \u003cem\u003eC. albicans \u003c/em\u003e(light blue circles) and enriched cultures\u003cem\u003e: A. fumigatus \u003c/em\u003esupplemented with hyphae secretome (green circles), \u003cem\u003eA. fumigatus \u003c/em\u003ewith yeast secretome (purples circles), \u003cem\u003eC. albicans \u003c/em\u003ewith hyphae secretome (\u003cem\u003eA. fumigatus\u003c/em\u003e) (pink circles) and \u003cem\u003eC. albicans\u003c/em\u003e with conidia secretome (yellow circles) after 5 days incubation at 25 °C. Coloured ellipses represent the 95% confidence regions of each clustering. \u003cstrong\u003eb \u003c/strong\u003eClustering analysis of the different metabolites in each group. Right\u003cstrong\u003e \u003c/strong\u003ecolumn of the heatmap\u003cstrong\u003e:\u003c/strong\u003e 27 significant metabolites identified with ANOVA test (p\u0026lt;0.01). Bottom of the heatmap: \u003cem\u003eC. albicans \u003c/em\u003eculture control in 3 replicates on the left vs. enriched cultures with \u003cem\u003eA. fumigatus \u003c/em\u003ehyphae (4 replicates) and enriched culture with conidia secretome (3 replicates) on the right. \u003csup\u003e1\u003c/sup\u003eH-NMR and timsTOF LC-MS data were combined and normalized with log transformation for the statistical analysis.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/5f2392ae0522ccb86fbf4e36.png"},{"id":107452867,"identity":"037db594-b091-4fe1-952c-22284a9299bc","added_by":"auto","created_at":"2026-04-21 15:30:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":301244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFungal secretomes induce different metabolite profile\u003c/strong\u003e. Volcano plots comparing metabolites abundance in enriched cultures vs. monocultures: \u003cstrong\u003ea \u003c/strong\u003e\u003cem\u003eC. albicans \u003c/em\u003ewith conidia secretome \u003cstrong\u003eb\u003c/strong\u003e \u003cem\u003eC. albicans \u003c/em\u003eand hyphae secretome \u003cstrong\u003ec \u003c/strong\u003e\u003cem\u003eA. fumigatus \u003c/em\u003eand yeast secretome\u003cstrong\u003e d\u003c/strong\u003e \u003cem\u003eA. fumigatus \u003c/em\u003eand hyphae secretome. Only significant metabolites are represented (\u003cem\u003ep-\u003c/em\u003evalue 0.05 FDR and fold change threshold 1.2). Positive log2 (fold change) values correspond with increased abundance in enriched culture and culture control (red) and negative log2 (fold change) values are the decrease of metabolite abundance. \u003csup\u003e1\u003c/sup\u003eH-NMR and LC-MS data were log-transformed and combined for statistical analysis.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/b111a046734d8ababbc69872.png"},{"id":107452866,"identity":"0271da3f-897c-49fb-b302-24f786b47e90","added_by":"auto","created_at":"2026-04-21 15:30:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67767,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDecrease of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emycotoxins in presence of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esecretomes. \u003c/strong\u003eMetabolic heatmap of \u003cem\u003eA. fumigatus \u003c/em\u003eculture supplemented with \u003cem\u003eC. albicans \u003c/em\u003esecretomes. Significant metabolite selection based on ANOVA (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) are listed on the right column of the heatmap. Scale bar: concentration value of the metabolites (red: highest concentration and blue: lowest concentration). Replicates of the different conditions are displayed on the bottom of the heatmap: \u003cem\u003eA. fumigatus \u003c/em\u003eculture on the left, then \u003cem\u003eA. fumigatus\u003c/em\u003e culture with \u003cem\u003eC. albicans\u003c/em\u003e hyphae secretome and \u003cem\u003eA. fumigatus \u003c/em\u003eculture with yeast secretome. Data was generated from timsTOF LC-MS analysis in Reversed Chromatography (RP) in both positive and negative ionization mode.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/a83f2eb6d830280c78c70b95.png"},{"id":107452870,"identity":"1d0c5ded-1d99-421b-99fa-f01081c98a2a","added_by":"auto","created_at":"2026-04-21 15:30:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":248036,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlteration of Pyrimidine and purine pathway in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. fumigatus \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eenriched culture. \u003c/strong\u003eEnrichment pathway analysis shows the most significantly altered metabolic pathways between \u003cem\u003eA. fumigatus \u003c/em\u003emonoculture and enriched culture \u003cstrong\u003ea \u003c/strong\u003eMetabolite set enrichment overview (top 25)\u003cstrong\u003e \u003c/strong\u003eof \u003cem\u003eA. fumigatus \u003c/em\u003ewith yeast secretome vs. monoculture (highest \u003cem\u003ep\u003c/em\u003e value in red: 0.003881 and yellow lowest \u003cem\u003ep\u003c/em\u003e=: 0.77297). \u003cstrong\u003eb\u003c/strong\u003e Significant metabolic set from purines nucleosides and nucleotides. \u003cstrong\u003ec\u003c/strong\u003e Significant metabolic set from pyrimidine nucleosides and nucleotides (black: monoculture and blue: enriched culture with yeast secretome) (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). \u003cstrong\u003ed \u003c/strong\u003eMetabolite set enrichment overview (top 25) of \u003cem\u003eA. fumigatus \u003c/em\u003ewith hyphae secretome vs. monoculture (highest \u003cem\u003ep\u003c/em\u003e value in red: 0.017477 and yellow lowest \u003cem\u003ep\u003c/em\u003e=0.62354 ). \u003cstrong\u003ee \u003c/strong\u003eSignificant metabolic set from purines nucleosides and nucleotides.\u003cstrong\u003e f \u003c/strong\u003eSignificant metabolic set from pyrimidine nucleosides and nucleotides (black: monoculture and blue: enriched culture with hyphe secretome) (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Boxplots show median and interquartile range (IQR); whiskers indicate the most extreme non-outlier values. Metabolite set library was based on chemical structures (main-class) on MetaboAnalyst. \u003csup\u003e1\u003c/sup\u003eH-NMR and timsTOF LC-MS data were combined and normalized with log transformation for the statistical analysis.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/7be6b8429fa8e85d4e85302e.png"},{"id":107489327,"identity":"97533ac9-9761-4d21-ac21-3677f4a7b003","added_by":"auto","created_at":"2026-04-22 02:47:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":334581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlteration of pyrimidine and purine pathway in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. albicans \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eenriched culture\u003c/strong\u003e. Enrichment pathway analysis shows the most significantly altered metabolic pathways between \u003cem\u003eC. albicans \u003c/em\u003emonoculture and enriched culture. \u003cstrong\u003ea\u003c/strong\u003e Metabolite set enrichment overview (top 25) of \u003cem\u003eC. albicans \u003c/em\u003ewith conidia secretome vs. monoculture (highest \u003cem\u003ep\u003c/em\u003e value in red: 3.49x10\u003csup\u003e-6\u003c/sup\u003e and yellow lowest \u003cem\u003ep\u003c/em\u003e= 0.96468). \u003cstrong\u003eb \u003c/strong\u003eSignificant metabolic set from purine nucleosides and nucleotides. \u003cstrong\u003ec \u003c/strong\u003eSignificant metabolic set from pyrimidine nucleosides and nucleotides (black: monoculture and blue: enriched culture with conidia). \u003cstrong\u003ed\u003c/strong\u003e Significant metabolic set from indoles and derivatives. \u003cstrong\u003ee\u003c/strong\u003e Metabolite set enrichment overview (top 25) of \u003cem\u003eC. albicans \u003c/em\u003ewith hyphae secretome vs. monoculture (highest \u003cem\u003ep\u003c/em\u003e value in red:0.0012 and yellow lowest \u003cem\u003ep\u003c/em\u003e=0.96106). \u003cstrong\u003ef \u003c/strong\u003eSignificant metabolic set from purine nucleosides and nucleotides. \u003cstrong\u003e\u0026nbsp;g \u003c/strong\u003eSignificant metabolic set from\u003cstrong\u003e \u003c/strong\u003epyrimidine nucleotides and nucleosides. \u003cstrong\u003eh \u003c/strong\u003eSignificant metabolic set from\u003cstrong\u003e \u003c/strong\u003eindoles and derivatives are represented (black: monoculture and blue: enriched culture with hyphae) (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). Boxplots show median and interquartile range (IQR); whiskers indicate the most extreme non-outlier values. Metabolite set library was based on chemical structures (main class) on MetaboAnalyst. \u003csup\u003e1\u003c/sup\u003eH-NMR and timsTOF LC-MS data were combined and normalized with log transformation for the statistical analysis.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/a8efe96621d036ec2fbb7ba9.png"},{"id":107452869,"identity":"26d72e0e-869e-456f-a7c2-21d3498449f7","added_by":"auto","created_at":"2026-04-21 15:30:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":700149,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphotype purity verification for secretome preparation\u003c/strong\u003e. \u003cstrong\u003ea \u003c/strong\u003epure conidial suspension (\u003cem\u003eA. fumigatus\u003c/em\u003e, 10⁶ conidia/mL) after 8 h in liquid AMM at 30 °C \u003cstrong\u003eb \u003c/strong\u003epure hyphal solution obtained after 12 h incubation (initial inoculum 10⁶ conidia/mL) at 37 °C in liquid RPMI to induce hyphae. \u003cstrong\u003ec \u003c/strong\u003epure yeast suspension grown in liquid YPD during 8 h at 30 °C \u003cstrong\u003ed\u003c/strong\u003e hyphae were obtained after 12 h incubation in liquid RPMI with low yeast inoculum (10⁶ yeast/mL) at 37 °C. Scale bar: 312.5 μm.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/7285c466385cc4a9335de294.png"},{"id":107490123,"identity":"36539fc9-766b-4693-8da1-0d13b9c97708","added_by":"auto","created_at":"2026-04-22 02:50:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3622605,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/b6a3c097-9192-4b47-98ea-6c3ab37df72b.pdf"},{"id":107489041,"identity":"32e7c7c4-92c3-4eca-9121-7d8bdbaec9bd","added_by":"auto","created_at":"2026-04-22 02:46:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3099337,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/369f2e049bda3a2dd7bcdbff.pdf"},{"id":107452871,"identity":"f4b41b2d-d2e6-49ce-aaf3-53a7b646aa04","added_by":"auto","created_at":"2026-04-21 15:30:46","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":155342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eUDP-GlcNAc:\u003c/strong\u003e Uridine diphosphate-N-acetylglucosamine and \u003cstrong\u003eUDP-glucose\u003c/strong\u003e: uridine diphosphate glucose). Scheme created with Biorender.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9059703/v1/030638e15b345087b43259a4.png"}],"financialInterests":"Competing interest reported. C.T. reports a research grant by Bruker Switzerland AG.","formattedTitle":"Elucidation of the secretory crosstalk between the pathogens Candida albicans and Aspergillus fumigatus via multimodal metabolomics","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe incidence of fungal superinfections is rising in both immunocompromised and immunocompetent patients. Among the fungal pathogens implicated in superinfections, \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e are the most prevalent worldwide and the primary causative agents of invasive fungal infections, particularly pulmonary infections and bloodstream infections\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. While the clinical burden of \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e coinfection is substantial, it remains often underrecognized. Both pathogens co-occur in patients with cystic fibrosis, invasive pulmonary aspergillosis complicated by candidiasis, or pleural infections\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. For example, in cystic fibrosis patients, fungal prevalence reaches 45.7% for \u003cem\u003eA. fumigatus\u003c/em\u003e and 75.5% for \u003cem\u003eC. albicans\u003c/em\u003e, with A. \u003cem\u003efumigatus\u003c/em\u003e coinfection occurring in 60% of cases alongside \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eaeruginosa\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Similarly, among COVID-19 patients, fungal infections are predominantly caused by \u003cem\u003eA. fumigatus\u003c/em\u003e (3.71%)\u003csup\u003e7\u003c/sup\u003e and \u003cem\u003eC. albicans\u003c/em\u003e (2.39%)\u003csup\u003e8\u0026ndash;11\u003c/sup\u003e. Critically, \u003cem\u003eC. albicans\u003c/em\u003e colonizes over 50% of patients asymptomatically\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e creating a reservoir for opportunistic invasion when immune defences are compromised.\u003c/p\u003e \u003cp\u003eDual fungal infections pose two interconnected threats that compromise patient survival. First, polymicrobial infections fundamentally alter disease dynamics compared to single-species infections\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The presence of one species may enhance the virulence, abundance, or persistence of others, establishing a self-reinforcing pathogenic cycle\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Second, with pathogens with high colonization potential, such as \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, there is a risk of persistent infections that are difficult to eradicate\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Together, these factors create a silent clinical crisis: two pathogens coexist and synergistically worsen patient prognosis while presenting as a single infection.\u003c/p\u003e \u003cp\u003eDespite the clinical urgency of dual fungal infections, fungal\u0026ndash;fungal interactions remain strikingly under-explored compared to other polymicrobial systems\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Bacteria\u0026ndash;bacteria, bacteria\u0026ndash;fungi and viruses-fungi pairs in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. For instance, \u003cem\u003eP. aeruginosa\u003c/em\u003e demonstrated complex interactions with both study fungi: it can both inhibit and promote \u003cem\u003eA. fumigatus\u003c/em\u003e growth in cystic fibrosis biofilm, while producing yeast-toxic pigments against \u003cem\u003eC. albicans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Conversely, \u003cem\u003eC. albicans\u003c/em\u003e colonization predisposes patients to \u003cem\u003eP. aeruginosa\u003c/em\u003e pneumonia, illustrating the clinical significance of these interactions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. An example of bacterial-fungal synergism is the capacity of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e to enhance \u003cem\u003eC. albicans\u003c/em\u003e adhesion to mucosal surfaces\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Additionally, viral infections, particularly COVID-19, predispose patients to \u003cem\u003eA. fumigatus\u003c/em\u003e superinfection through epithelial damage, facilitating conidia internalization into cells\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn contrast, fungal\u0026ndash;fungal interactions, particularly between opportunistic pathogens, remain largely unexplored. While one study reported fungistatic effects of \u003cem\u003eC. albicans\u003c/em\u003e against \u003cem\u003eA. fumigatus in vitro\u003c/em\u003e, the underlying mechanisms were not elucidated\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This knowledge gap is clinically significant, as polymicrobial infections involving fungal agents are challenging to diagnose and treat, often resulting in worse patient outcomes and increased mortality rates. Given their high clinical relevance and frequent co-occurrence, understanding the crosstalk between \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e is essential for elucidating the dynamics of coinfection and the factors enabling fungal persistence.\u003c/p\u003e \u003cp\u003eHere, we investigated the interactions between \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans in vitro\u003c/em\u003e using enriched cultures experiments designed to mimic dual fungal infection. To achieve this aim, we developed a secretome-metabolite exchange analysis approach using combinatory multimodal metabolomics analysis by targeted proton nuclear magnetic resonance spectroscopy (\u003csup\u003e1\u003c/sup\u003eH-NMR) and untargeted liquid chromatography (LC) trapped ion mobility spectrometry time-of-flight mass spectrometry (timsTOF LC-MS) using large CCS (collision cross section) libraries for maximum annotation coverage. To explore the role of different morphological forms in metabolite production, we studied the metabolic interaction between whole \u003cem\u003eA. fumigatus\u003c/em\u003e culture with yeast or hyphae secretomes as well as \u003cem\u003eC. albicans\u003c/em\u003e culture exposed to \u003cem\u003eA. fumigatus\u003c/em\u003e secretome (conidia or hyphae). Using this multimodal targeted and untargeted approach of two state of the art metabolomics technologies enabled us to identify antagonistic behaviours and potential mutualistic mechanisms that could explain the severity and persistence of dual fungal infections in vulnerable patient populations, paving the ground for improved future target discovery and treatments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eCoculture of\u003c/b\u003e \u003cb\u003eA. fumigatus\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003eshows interaction at the hyphal front\u003c/b\u003e. To characterize secretory metabolic interactions between \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, we first performed enrichment culture experiments using secretomes from different fungal morphotypes (yeast, conidia and hyphae) added to the whole fungal culture and analysed the samples by both \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-NMR and timsTOF LC-MS. Our multimodal metabolomics approach could highlight differential metabolite signatures between enriched culture and culture control conditions. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea shows the workflow for the analysis of metabolites in enriched cultures: \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e cultures supplemented with secretomes were grown during 5 days at 25\u0026deg;C. The monoculture control (containing only \u003cem\u003eA. fumigatus\u003c/em\u003e or only \u003cem\u003eC. albicans)\u003c/em\u003e were grown under the same conditions. Enriched cultures and monoculture were extracted with organic solvents for H\u0026sup1;-NMR and timsTOF LC-MS measurements.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2\u003c/b\u003e: \u003cb\u003eA. fumigatus\u003c/b\u003e \u003cb\u003econidia secretome leads to shift in metabolites distribution. a\u003c/b\u003e Discriminant analysis score plot of metabolites obtained from pure culture \u003cem\u003eA. fumigatus\u003c/em\u003e (red circles) and \u003cem\u003eC. albicans\u003c/em\u003e (light blue circles) and enriched cultures: \u003cem\u003eA. fumigatus\u003c/em\u003e supplemented with hyphae secretome (green circles), \u003cem\u003eA. fumigatus\u003c/em\u003e with yeast secretome (purples circles), \u003cem\u003eC. albicans\u003c/em\u003e with hyphae secretome (\u003cem\u003eA. fumigatus\u003c/em\u003e) (pink circles) and \u003cem\u003eC. albicans\u003c/em\u003e with conidia secretome (yellow circles) after 5 days incubation at 25\u0026deg;C. Coloured ellipses represent the 95% confidence regions of each clustering. \u003cb\u003eb\u003c/b\u003e Clustering analysis of the different metabolites in each group. Right column of the heatmap: 27 significant metabolites identified with ANOVA test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Bottom of the heatmap: \u003cem\u003eC. albicans\u003c/em\u003e culture control in 3 replicates on the left vs. enriched cultures with \u003cem\u003eA. fumigatus\u003c/em\u003e hyphae (4 replicates) and enriched culture with conidia secretome (3 replicates) on the right. \u003csup\u003e1\u003c/sup\u003eH-NMR and timsTOF LC-MS data were combined and normalized with log transformation for the statistical analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eH\u0026sup1;-NMR and timsTOF LC-MS highlight the impact of\u003c/b\u003e \u003cb\u003eA. fumigatus\u003c/b\u003e \u003cb\u003econidia on\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003emetabolic activity\u003c/b\u003e. H\u0026sup1;-NMR and timsTOF LC-MS analysis were conducted to explore the metabolic interactions between \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eA. fumigatus\u003c/em\u003e after 5 days of enriched cultures compared to the monoculture. Metabolites of interest are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (\u003cem\u003eC. albicans\u003c/em\u003e metabolites linked to virulence), Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (\u003cem\u003eA. fumigatus\u003c/em\u003e mycotoxins) and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (metabolites common to both fungi) and their known biological functions described.\u003c/p\u003e \u003cp\u003eTo determine the degree of difference between the metabolite profile observed during enriched culture and monoculture, Principal Component Analysis (PCA) scores were generated based on combined H\u0026sup1;-NMR and timsTOF LC-MS measurements (Fig.\u0026nbsp;2a\u003cb\u003e).\u003c/b\u003e PCA was applied to all data sets: \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e monocultures and enriched culture. PCA scores resulted in three distinct clusters, where the \u003cem\u003eC. albicans\u003c/em\u003e culture enriched with conidia secretome was separated from the other groups. There was no difference between the \u003cem\u003eC. albicans\u003c/em\u003e monoculture and the \u003cem\u003eC. albicans\u003c/em\u003e culture supplemented with \u003cem\u003eA. fumigatus\u003c/em\u003e hyphae. \u003cem\u003eA. fumigatus\u003c/em\u003e monoculture, \u003cem\u003eA. fumigatus\u003c/em\u003e culture supplemented with \u003cem\u003eC. albicans\u003c/em\u003e hyphae or yeast were grouped together (Fig.\u0026nbsp;2a). Secretome from conidia seems to induce a unique metabolome profile on \u003cem\u003eC. albicans\u003c/em\u003e culture compared to the other conditions.\u003c/p\u003e \u003cp\u003eSince our PCA analysis revealed a distinct clustering of the enriched culture with conidia secretome compared to the other conditions, we next generated a metabolic heatmap to visualize metabolite-level differences driving this separation. The metabolic heatmap generated 27 significant metabolites through ANOVA analysis (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) from combined H\u0026sup1;-NMR and timsTOF LC-MS data sets (Fig.\u0026nbsp;2b). The metabolite distribution in the \u003cem\u003eC. albicans\u003c/em\u003e culture control remained unchanged when \u003cem\u003eA. fumigatus\u003c/em\u003e hyphae secretome was added. Among the 27 significant metabolites identified, 13 metabolites were increased in both conditions (control and \u003cem\u003eC. albicans\u003c/em\u003e enriched culture with \u003cem\u003eA. fumigatus\u003c/em\u003e hyphae secretome): 5-ethyl-2.4-dimethyloxazole, n-isopropyl-l-glutamine, lactose, lactate, n6-acetyl-l-lysine myo-inositol, hypotaurine, pantothenic acid (Vitamin B5), lysine, (2e)-oct-2-enediol, butyrylcarnitine, penitricin D. The 14 other metabolites identified (o-acetylleucine, 10-oxodihydrobotry, waraterpol, 5-l-glutamyl-l-leucine, campyrone c, n-acetylleucine, l-norleucine, taurine, isoleucine, 3-(hydroxyacetyl)-indole, citrate and guanine were decrease. In contrast, adding conidia secretome to \u003cem\u003eC. albicans\u003c/em\u003e culture induced a switch in the metabolite distribution: the 13 metabolites that were elevated in the culture control and in presence of hyphae secretome became decreased while the 14 metabolites that were decreased with hyphae secretome and in the culture control became increased. Interestingly, virulent linked metabolites: n6-acetyl-l-lysine, a N-acyl-alpha amino acids, pantothenic acid and lysine (metabolite-biofilm related) were decreased in presence of conidia secretome\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Although, compounds responsible for remodelling \u003cem\u003eC. albicans\u003c/em\u003e cell wall architecture (lactose and lactate) were decreased in presence of conidia secretome. These findings, indicates that conidia secretome may induce changes in \u003cem\u003eC. albicans\u003c/em\u003e cell wall architecture (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlso, this conidia-specific metabolic reversal was reflected in the PCA analysis, where conidia secretome-enriched cultures clustered distinctly apart from both control and hyphae secretome conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cdiv description=\"\" class=\"Drawing\" id=\"4\" name=\"Grafik 4\"\u003e\u003c/div\u003e\u003c/h2\u003e \u003cp\u003e \u003cb\u003eFungal morphology-specific secretomes induce singular variation in metabolite distribution.\u003c/b\u003e Next, we used a volcano plot using (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to highlight individuals features presenting a significantly different change between the monoculture control and the enriched culture with secretomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the \u003cem\u003eC. albicans\u003c/em\u003e enriched culture with conidia secretome, 26 metabolites were significantly altered (10 metabolites were decreased and 16 metabolites increased) compared to 11 metabolites (7 metabolites decreased and 4 metabolites increased) for the \u003cem\u003eC. albicans\u003c/em\u003e enriched culture with hyphae secretome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Notably, trehalose, a metabolite produced from mannitol (trehalose biosynthesis pathway: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/33511160/#\u0026amp;gid=article-figures\u0026amp;pid=figure-3-uid-2\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/33511160/#\u0026amp;gid=article-figures\u0026amp;pid=figure-3-uid-2\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and glutathione, an antioxidant, were in lower concentration in presence of \u003cem\u003eA. fumigatus\u003c/em\u003e hyphae secretome in the \u003cem\u003eC. albicans\u003c/em\u003e culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). As seen in the heatmap (Fig.\u0026nbsp;2b), guanine, a nitrogenous base, was increased in presence of conidia and hyphae secretome. Amino acids, such as histidine and lysine were decreased in presence of conidia secretome while isoleucine, beta-alanine and L-norleucine were increased. Speradine B, an alkaloid already isolated from a sponge derived fungus \u003cem\u003eA. flavus\u003c/em\u003e MXH-X104 was only present when adding conidia secretome to the \u003cem\u003eC. albicans\u003c/em\u003e culture\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. fumigatus\u003c/em\u003e enriched culture supplemented with \u003cem\u003eC. albicans\u003c/em\u003e secretome displayed a different metabolites distribution compared to the \u003cem\u003eC. albicans\u003c/em\u003e enriched culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Metabolites involved in purine and pyrimidine pathway: inosine monophosphate (IMP), inosine were increased. The vitamin B3 (Niacinamide) was decreased in presence of yeast secretome in \u003cem\u003eA. fumigatus\u003c/em\u003e culture. Similarly, IMP, inosine and uridine-5\u0026rsquo;-monophosphate (UMP) level were increased by hyphae secretome. Interestingly, spirotryprostatin A, an \u003cem\u003eA. fumigatus\u003c/em\u003e mycotoxin was decreased in presence of \u003cem\u003eC. albicans\u003c/em\u003e hyphae secretome. These results indicate that fungal secretome added to the whole culture (\u003cem\u003eA. fumigatus\u003c/em\u003e or \u003cem\u003eC. albicans\u003c/em\u003e) induce unique metabolomic signature. Conidia secretome seemed to have the most impact on \u003cem\u003eC. albicans\u003c/em\u003e culture, since a larger group of metabolites were altered in the enriched culture compared to the other conditions. Carbon markers such as trehalose and glutathione were affected by \u003cem\u003eA. fumigatus\u003c/em\u003e hyphae secretome in \u003cem\u003eC. albicans\u003c/em\u003e culture.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibition of\u003c/b\u003e \u003cb\u003eA\u003c/b\u003e. \u003cb\u003efumigatus\u003c/b\u003e \u003cb\u003emycotoxins by\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e \u003cb\u003esecretome\u003c/b\u003e. The timsTOF LC-MS analysis detected 10 \u003cem\u003eAspergillus\u003c/em\u003e mycotoxins main virulence factors: Sphingofungins B and D, cyclotryprostatins A, spirotryprostatin A, TR-2 belonging to the fumitremorgins family, isomeric quinazoline-containing indole alkaloids such as fumiquinazoline F and D, aculeatsquinone D, alternariol 4-methyl-10-acetyl ether and speradine B (detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate how mycotoxins were distributed between culture conditions, a metabolic heatmap was generated from timsTOF LC-MS metabolomic data comparing \u003cem\u003eA. fumigatus\u003c/em\u003e pure culture with enriched cultures containing hyphae and yeast secretome (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among the 10 mycotoxins identified, three toxins: Spirotryprostatin A, sphingofungin B and D were significantly decreased in presence of \u003cem\u003eC. albicans\u003c/em\u003e secretomes. Interestingly, fumiquinazoline F and D, TR-2, aculeatsquinone D, alternariol 4-methyl-10-acetyl ether, speradine B and cyclotryprostatins A levels were comparable in all conditions (culture control and enriched cultures). Thus, molecules present in \u003cem\u003eC. albicans\u003c/em\u003e secretome may counteract certain mycotoxins produced by \u003cem\u003eA. fumigatus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAlteration of purine and pyrimidine pathways and cell wall precursors in enriched culture.\u003c/b\u003e Enrichment analysis was performed to see which metabolic pathways were the most active in the metabolites produced by the different fungal forms. Among the main class of metabolites specific to \u003cem\u003eA. fumigatus\u003c/em\u003e genus, we could identify five significant classes of metabolites: organooxygen compounds, carboxylic acids and derivatives, organonitrogen compounds, purine and pyrimidine pathway. Significant metabolites belonging to purine and pyrimidine pathways were produced in both \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e enriched cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, d and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, e). These specific pathways are essential in both species for virulence and growth\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34 CR35\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the purine pathway, the presence of yeast secretome in \u003cem\u003eA. fumigatus\u003c/em\u003e culture showed a variation of metabolites concentration belonging to the salvage pathway: IMP (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0025) and inosine (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0116) were significantly increased whereas adenosine monophosphate (AMP) (\u003cem\u003ede novo\u003c/em\u003e purine pathway) was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Hyphae secretome showed the same variation of metabolites in \u003cem\u003eA. fumigatus\u003c/em\u003e culture: significant increase of inosine (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0175) and IMP (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0104). The other metabolites stayed at the same level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). The purine pathway biosynthesis steps for fungal pathogens are visible here: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pmc.ncbi.nlm.nih.gov/articles/PMC5488104/figure/microorganisms-05-00033-f001/\u003c/span\u003e\u003cspan address=\"https://pmc.ncbi.nlm.nih.gov/articles/PMC5488104/figure/microorganisms-05-00033-f001/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eIn the pyrimidine pathway (for \u003cem\u003eA. fumigatus\u003c/em\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.researchgate.net/publication/340602879/figure/fig1/AS:11431281432025133@1746830936226/The-de-novo-pyrimidine-biosynthesis-pathway-which-leads-to-the-formation-of-UMP-In.tif\u003c/span\u003e\u003cspan address=\"https://www.researchgate.net/publication/340602879/figure/fig1/AS:11431281432025133@1746830936226/The-de-novo-pyrimidine-biosynthesis-pathway-which-leads-to-the-formation-of-UMP-In.tif\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), both yeast and hyphae secretome added to \u003cem\u003eA. fumigatus\u003c/em\u003e culture similarly increased UMP (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0318) and decreased uridine diphosphate-N-acetylglucosamine (UDP-N-acetylglucosamine) (precursor of chitin, a component of fungi cell wall). The other metabolites linked to pyrimidine pathway stayed at the same level: uridine diphosphate glucose (UDP-glucose, precursor of β-glucans, cell wall component) and uridine diphosphate-galactose (UDP-galactose) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. fumigatus\u003c/em\u003e secretome added to \u003cem\u003eC. albicans\u003c/em\u003e culture showed a different variation of metabolites from purine pathway compared to \u003cem\u003eC albicans\u003c/em\u003e enriched culture with \u003cem\u003eA. fumigatus\u003c/em\u003e secretome. ADP (salvage pathway) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00248) and 2\u0026rsquo;-O-methyladenosine (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00297) were significantly increased while AMP (\u003cem\u003ede novo\u003c/em\u003e purine pathway) and inosine (salvage pathway) were decreased by conidia secretome (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Hyphae secretome showed the same increased of compounds as with conidia secretome belonging to the salvage pathway: ADP, adenosine (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0171) and IMP. 2\u0026rsquo;-O-methyladenosine while AMP and inosine abundance did not change (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eIn pyrimidine pathway, both conidia and hyphae secretome decreased both \u003cem\u003eA. fumigatus\u003c/em\u003e conidia and hyphae secretome decreased UDP-N-acetylglucosamine as observed in \u003cem\u003eA. fumigatus\u003c/em\u003e enriched culture. UDP-galactose was significantly increase in \u003cem\u003eC. albicans\u003c/em\u003e enriched culture with hyphae secretome. UMP and UDP-glucose abundance was unchanged compared to the culture control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, g). Steps of \u003cem\u003eC. albicans\u003c/em\u003e pyrimidine pathway are visible here: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kegg.jp/pathway/cal00240\u003c/span\u003e\u003cspan address=\"https://www.kegg.jp/pathway/cal00240\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e showed different variation of metabolites belonging to purine and pyrimidine pathway that could be caused by stress. Cell wall components, especially UDP-N-acetylglucosamine was affected by all fungal secretome in enriched cultures.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSelective induction of indole pathway metabolites in\u003c/b\u003e \u003cb\u003eC. albicans\u003c/b\u003e culture enriched with \u003cb\u003eA. fumigatus\u003c/b\u003e \u003cb\u003esecretome\u003c/b\u003e. Key indole metabolites were significantly identified in \u003cem\u003eC. albicans\u003c/em\u003e enriched culture but were absent in enriched \u003cem\u003eA. fumigatus\u003c/em\u003e culture: tryptophol, indoleacetaldehyde, indoleacrylic acid, 5-hydroxytryptophol, indole-3-carboxaldehyde, 5-hydroxy-L-tryptophan and L-tryptophan (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, h). Indoles metabolites in \u003cem\u003eC. albicans\u003c/em\u003e may be involved in the synthesis of quorum sensing molecule, here, tryptophol, to regulate the population in response to stress (insufficient nutrients available). Tryptophol level was decreased in presence of conidia secretome (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Overall, the metabolites associated with indoles derivatives pathway were significantly decreased in presence of conidia secretome significantly decreased L-tryptophan (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00249), indoleacrylic acid (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0149) and 5-hydroxy-L-tryptophan (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00195), while indole-3-carboxaldehyde was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0294). Hyphae secretome decreased as well significantly indoleacetaldehyde (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00613) and 5-Hydroxy-L-tryptophan but the other metabolites stayed at the same level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). The presence of indole metabolites in \u003cem\u003eC. albicans\u003c/em\u003e enriched culture is specific to the production of quorum sensing molecule (tryptophol) in response to the presence of \u003cem\u003eA. fumigatus\u003c/em\u003e secretome.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStress resistance is modulated by carbon adaption.\u003c/b\u003e Organooxygen metabolites essential for fungal energetic metabolism were expressed in \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e enriched cultures (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). In \u003cem\u003eA. fumigatus\u003c/em\u003e enriched culture, the level of glycerol in presence of yeast secretome was significantly increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0121) while D-glucose level was unchanged. In presence of hyphae secretome, glycerol level was unchanged, and D-glucose was slightly increased. There was no trehalose detected whether yeast or hyphae secretome was added (\u003cb\u003eSupplementary Fig.\u0026nbsp;2a).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe distribution of metabolites belonging to organooxygen pathway for the \u003cem\u003eC. albicans\u003c/em\u003e culture supplemented with \u003cem\u003eA. fumigatus\u003c/em\u003e secretome was different (\u003cb\u003eSupplementary Fig.\u0026nbsp;2b\u003c/b\u003e). Organooxygen metabolites plays an important role in \u003cem\u003eC. albicans\u003c/em\u003e cell wall architecture. Glycerol level was decreased in presence of conidia secretome while D-glucose and trehalose were increased. Enriched culture with hyphae secretome showed different variation of these metabolites: there was a significant decrease of trehalose (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0112). D-glucose was slightly increased while glycerol was decreased. Variation of these carbon-linked metabolites may have induced structural modifications in \u003cem\u003eC. albicans\u003c/em\u003e cell wall upon exposure to \u003cem\u003eA. fumigatus\u003c/em\u003e secretomes. These variations in cell wall structure could impact fungal response to stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe nature of polymicrobial interaction \u0026mdash;whether competitive, antagonistic, or mutualistic\u0026mdash; critically influences disease severity and patient outcome. Our study provides the first multimodal metabolomics-based characterization of the interaction between \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e revealing antagonistic behaviour and raising the possibility of mutualistic coexistence. This dual interaction paradigm has direct clinical implications for management of immunocompromised patients, where both pathogens may co-occur.\u003c/p\u003e \u003cp\u003eWe chose to focus on secretomes in our enriched cultures experiment to specifically capture the metabolites released by distinct fungal morphological forms. This approach directly models the clinical scenario: during infection, the host is simultaneously exposed to multiple fungal morphotypes \u0026mdash;yeast, conidia, and hyphae\u0026mdash; which coexist and release different repertoires of metabolites\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Understanding these morphotype-specific contributions is essential for predicting disease progression, as each form may differentially modulate the coculture environment and host response.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. albicans\u003c/em\u003e culture supplemented with conidia secretome formed a distinct metabolite cluster reflecting a dormant state, characterized by low respiratory activity, reduced amino acid levels, and trehalose storage\u0026mdash;typically metabolized during germination\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. By contrast, \u003cem\u003eC. albicans\u003c/em\u003e culture enriched with hyphal secretome displayed metabolic activation with increased lactate and amino acid biosynthesis, consistent with the energy demands of rapid growth and secondary metabolite production\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMetabolite levels linked to pyrimidine and purine pathway varied under enriched cultures compared with the pure culture for all conditions suggesting metabolic stress. This represents the first layer of antagonism: competition for shared resources in the medium. The metabolite profiles could have indicated a shift toward \u003cem\u003ede novo\u003c/em\u003e biosynthesis of purines and pyrimidines\u0026mdash;a less energy-efficient strategy typically triggered by nutrient limitation. Specifically, UMP (\u003cem\u003ede novo\u003c/em\u003e pyrimidine pathway) was increased while UDP-N-acetylglucosamine, essential for cell wall construction in both fungi, was decreased\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. In the metabolites belonging to the \u003cem\u003ede novo\u003c/em\u003e purine pathway: AMP level was reduced and ADP and IMP increased, reflecting altered energy status and metabolic flux through this biosynthetic route. Together, these changes suggest that resource competition forces both fungi to rely on metabolically costly \u003cem\u003ede novo\u003c/em\u003e synthesis pathways for survival\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBeyond metabolic competition, a second layer of antagonism emerged: a direct chemical warfare through secreted antifungal compounds. On solid media, \u003cem\u003eA. fumigatus\u003c/em\u003e growth was restricted by \u003cem\u003eC. albicans\u003c/em\u003e, producing a \u0026ldquo;distance-inhibition\u0026rdquo; zone with reduced hyphal density\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. This pattern, consistent with prior observations of inhibitory activity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, suggests active release of antifungal molecules, which our metabolomics approach confirmed through identification of tryptophol. Tryptophol is a quorum-sensing molecule produced by \u003cem\u003eC. albicans\u003c/em\u003e that represses hyphal development and inhibits both fungi\u003csup\u003e\u003cspan additionalcitationids=\"CR48 CR49\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The growth limitation of \u003cem\u003eA. fumigatus\u003c/em\u003e thus results from a dual mechanism: (i) metabolic stress from nutrient competition forcing switch to energy-expensive \u003cem\u003ede novo\u003c/em\u003e biosynthesis, and (ii) direct chemical inhibition through tryptophol release.\u003c/p\u003e \u003cp\u003eConversely, \u003cem\u003eA. fumigatus\u003c/em\u003e secreted mycotoxins with antifungal activity against \u003cem\u003eC. albicans\u003c/em\u003e, especially sphingofungins B, Fumiquinalozine D and spirotryprostatin A. Sphingofungins disrupt sphingolipids biosynthesis in the fungal cell wall\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, while spirotryprostatin A exerts antimitotic and antimicrobial activity\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Interestingly, both mycotoxins (spirotryprostatin A and sphingofungin B) were reduced in enriched culture with \u003cem\u003eC. albicans\u003c/em\u003e secretome (yeast and hyphae), suggesting possible metabolic counteractions by \u003cem\u003eC. albicans.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eA critical clinical concern emerged from our data: antagonistic fungal interactions generated metabolites that may directly worsen patient outcomes. We identified indole-derived metabolites, particularly indoleacrylic acid, indole-3-acetaldehyde, and indole-3-carboxyaldehyde released in \u003cem\u003eC. albicans\u003c/em\u003e culture supplemented with \u003cem\u003eA. fumigatus\u003c/em\u003e secretome. These metabolites may be part of tryptophan-independent pathway, which exist in fungi species and may be expressed in yeast strains\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Recent evidence linked these metabolites to severe clinical outcomes in pneumonia\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Specifically, indole-3-acetaldehyde (which converts to indole-3-acetic acid), and its derivative indole-3-acetic acid have been associated with increased pneumonia severity (higher pulmonary damages, production of reactive oxygen species, increase of immune resistance), higher hospitalization risk, and elevated mortality rates\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Given that ventilator-associated pneumonia and bloodstream infections are the predominant intensive care unit (ICU)-acquired infections in immunocompromised patients mainly caused by \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e and the production of indole metabolites during fungal interactions may significantly exacerbate disease severity and contribute to poor clinical outcomes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough antagonistic competition dominates the metabolic landscape, our data revealed mechanisms by which these pathogens may simultaneously support each other. The detected \u003cem\u003eA. fumigatus\u003c/em\u003e mycotoxins fumiquinalozines A and cycloprostatin A are known to be associated with biofilm formation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Their unchanged levels in enriched culture with the \u003cem\u003eC. albicans\u003c/em\u003e secretome could suggests that \u003cem\u003eA. fumigatus\u003c/em\u003e can maintain biofilm formation in the presence of the yeast. DL-pipecolate, a metabolite involved in \u003cem\u003eC. albicans\u003c/em\u003e biofilm remained also unchanged in presence of \u003cem\u003eA. fumigatus\u003c/em\u003e secretome\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The previously indole metabolite involved in pneumonia exacerbation: indole-3-acetaldehyde which converts to indole-3-acetic acid, is also known to promote fungi filamentation and biofilm\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Despite \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e having distinct biofilms\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, both fungi produce biofilms rich in lipids and extracellular DNA and share the same chitin precursor (UDP-N-acetylglucosamine) and β-glucans precursor (UDP-glucose)\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, which may enable structural interconnection, providing a potential mechanism by which \u003cem\u003eC. albicans\u003c/em\u003e stabilizes or enhances \u003cem\u003eA. fumigatus\u003c/em\u003e biofilm formation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Mixed biofilms are common in polymicrobial infections and contribute to pathogens persistence through combined protective matrix and antifungal resistance\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Some mycotoxins identified in this study are established tissue-damaging agents that compromise pulmonary tissue integrity\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e creating a disrupted host niche conducive to enhanced colonization and modify disease progression. Together, mycotoxin-induced tissue damage and mixed biofilm-mediated persistence enable synergistic interactions that may significantly exacerbate infection severity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile our study highlighted antagonistic behaviour between \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e, co-infections pathogens usually interact synergistically and support each other\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Since \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eA. fumigatus\u003c/em\u003e tolerated each other in the same environment (in solid medium), mutualism between the two species could also exist despite their antagonistic behaviour. To examine this possibility, we recorded a 12 h liquid coculture in a slide chamber and confirmed that both fungi remained viable. The \u003cem\u003eC. albicans\u003c/em\u003e inoculum increased considerably in the presence of \u003cem\u003eA. fumigatus\u003c/em\u003e, and both species formed hyphae and pseudohyphae. We also observed the formation of multicellular clusters from \u003cem\u003eC. albicans\u003c/em\u003e in the presence of \u003cem\u003eA. fumigatus\u003c/em\u003e, a virulence-associated stress response\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). This experimental evidence is supported by clinical observations: Kubota \u003cem\u003eet al.\u003c/em\u003e, (2023) reported invasive pulmonary aspergillosis combined with candidiasis in an immunocompromised patient, where yeasts were embedded within \u003cem\u003eA. fumigatus\u003c/em\u003e hyphal networks\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The embedding of yeast cells within the hyphae suggests a mutualistic interaction, highlighting that these two fungi can coexist and adapt to one another in the same niche.\u003c/p\u003e \u003cp\u003eAlthough this work significantly advances understanding of \u003cem\u003eA. fumigatus\u003c/em\u003e - \u003cem\u003eC. albicans\u003c/em\u003e interactions, it is not without limitations. The metabolomic measurements of secretomes added to enriched cultures were excluded because the metabolite profiles were inconsistent or of poor quality. We employed three different culture media with distinct purposes: AMM and YPD to maximize metabolite production in enriched cultures, and PDA as a neutral basal medium for fungal coculture. Metabolite profiles in some secretome conditions showed minimal differences from monocultures, likely due to lower secretome concentration.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eUsing a novel multimodal metabolomics approach, this study demonstrates that \u003cem\u003eA. fumigatus\u003c/em\u003e and \u003cem\u003eC. albicans\u003c/em\u003e engage in complex secretory metabolic interactions characterized by both potential antagonism and mutualism. For immunocompromised populations, where these fungi represent the predominant invasive pathogens, their capacity for coexistence presents a critical clinical threat. Colonization by one species creates a permissive niche for the other; compounding this risk, the production of tissue-damaging mycotoxins, inflammatory indole metabolites that exacerbate pneumonia, and mixed biofilms collectively drive severe, treatment-resistant infections. The vulnerability of immunocompromised patients is further heightened by the clinically indistinguishable symptoms of mono- and polymicrobial infections, which impede accurate diagnosis and delay appropriate dual-species therapy, ultimately worsening prognosis and increasing mortality. Understanding these metabolic mechanisms is essential for developing targeted therapeutic strategies that disrupt fungal interactions and improve outcomes in vulnerable populations. Future studies in clinical settings mimicking coinfection are needed to elucidate mutualism mechanisms, particularly the role of mixed biofilm formation and dosage-dependent functional impact of metabolites.\u003c/p\u003e"},{"header":"METHOD details","content":"\u003cp\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eC. albicans\u003c/strong\u003e \u003cstrong\u003estrains and growth conditions.\u003c/strong\u003e \u003cem\u003eA. fumigatus\u003c/em\u003e wild-type strain ATCC 46645 and \u003cem\u003eC. albicans\u003c/em\u003e SC5314 (kindly sent by the University Bourgogne Franche-Comte in Dijon) were used in this work. For the culture control, \u003cem\u003eA. fumigatus\u003c/em\u003e was grown on \u003cem\u003eAspergillus\u003c/em\u003e minimal media (AMM) and \u003cem\u003eC. albicans\u003c/em\u003e on yeast peptone dextrose broth (YPD) (Sigma-Aldrich, Damstadt, Germany) (500 \\(\\mu\\)L of 10\u003csup\u003e6\u003c/sup\u003e cells/mL spread on Petri dish for both fungi). Enriched cultures were grown in the same conditions supplemented with secretome for the\u0026nbsp;\u003csup\u003e1\u003c/sup\u003eH-NMR and TimsTOF LC-MS analysis. Coculture containing both whole strains were grown on Potato dextrose agar extract (PDA) (Sigma-Aldrich, Damstadt, Germany) 10 days at 25°C to allow complete growth of both fungi and have fungal morphological pattern.\u003cbr\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e \u003cstrong\u003esecretomes.\u003c/strong\u003e Conidia suspensions were obtained by harvesting grown mycelia on AMM plates with phosphate buffer saline PBS-T (DPBS, Gibco Life technologies, Carlsbad, CA, USA) (Tween 20 0.01%) followed by filtration through 100 µm and 30 µm filters (Miltenyi Biotec, Bergisch Gladbach, Germany). For conidia secretome preparation, 10⁶ conidia/mL were incubated for 6–8 h at 30°C. Cultures were then centrifuged centrifuged at 3,000 × g for 10 min at room temperature (RT), and supernatants were collected and filtered (100 µm and 30 µm) to remove residual conidia. For\u0026nbsp;\u003cem\u003eA. fumigatus\u003c/em\u003e hyphal secretome, 10⁶ conidia/mL were inoculated in RPMI medium and incubated at 37°C for 16–18 h to allow complete hyphal development. Supernatants were collected by centrifugation at centrifuged at 3,000 × g for 10 min at RT and filtered as described above.\u003cbr\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e \u003cstrong\u003esecretomes.\u003c/strong\u003e For yeast secretome, yeasts were collected from an overnight culture, washed twice with YPD (4,600 × g for 3 min at RT), and adjusted to 10⁶ cells/mL (OD₆₀₀). Yeast suspension was incubated during 6h-8h at 30°C to generate metabolites. For hyphal secretome, yeast cells at 10⁶ cells/mL were incubated at 37°C for 16–18 h in RPMI to induce hyphal development. Both secretomes were then centrifuged at 3,000 × g for 10 min at RT, and supernatants were collected and sequentially filtered (100 µm and 30 µm) to remove residual cells.\u003c/p\u003e\n\u003cp\u003eTo ensure secretome purity, cultures were microscopically examined before centrifugation to confirm the presence of a single morphological form (100% yeast, conidia, or hyphae). Only cultures displaying 100% morphotype homogeneity were retained for secretome collection. Following centrifugation, supernatants were additionally filtered if any residual cells were detected. Representative microscopy images confirming secretome purity and morphotype specificity are provided in (Fig.\u0026nbsp;7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnriched cultures of\u003c/strong\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e. To assess the impact of secretomes on fungal metabolism, enriched cultures were prepared by adding secretomes to pre-established cultures. For enriched \u003cem\u003eA. fumigatus\u003c/em\u003e cultures, 10⁶ conidia/mL were pre-grown on Petri dishes for 1 day at 37°C to allow initial growth, then 800 µL of \u003cem\u003eC. albicans\u003c/em\u003e secretome (yeast or hyphal) was added. For enriched \u003cem\u003eC. albicans\u003c/em\u003e cultures, 10⁶ yeast cells/mL were pre-grown for 1 day at 30°C, then 800 µL of \u003cem\u003eA. fumigatus\u003c/em\u003e secretome (conidial or hyphal) was added. All plates (monocultures and enriched cultures) were subsequently incubated for 5 days at 25°C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. fumigatus\u003c/strong\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cstrong\u003eC. albicans\u003c/strong\u003e\u003cstrong\u003ecoculture\u003c/strong\u003e. To observe fungal morphological pattern, solid coculture using both fungi was performed. 100 µL of each species was point inoculated on the extremity of the Petri dish at 1x10\u003csup\u003e6\u003c/sup\u003e cells/mL. The cultures were incubated 10 days at 25°C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSamples collection procedure.\u003c/strong\u003e The enriched cultures and monocultures were rinse two times with 5 mL pre-cooled (4°C) PBS. PBS was removed quickly, and samples collected into 15 ml falcon tubes and pour quickly in liquid nitrogen. Samples were then directly transferred to ice. 0.8 mL of pre-chilled at -80°C methanol was added to the falcon tubes. The samples were vortex strongly for 10 sec and placed on dry ice. All sample were stored at -80°C until measurements. Only the cell pellets were used for multimodal metabolomics analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolites extraction and\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eH-NMR analysis\u003c/strong\u003e. The extracts were transferred into 2 mL\u003c/p\u003e\n\u003cp\u003eAFA™ Covaris glass tubes. Each sample was thoroughly mixed by vortexing with 900 µL methanol, 100 µL chloroform, and 100 µL ultrapure water before being loaded in the Covaris ultrasonicator E220 Evolution. Following extraction, solutions were loaded into a centrifuge (12,000g force, 30 min at 6°C) for complete phase separation. Centrifuged samples were later manually collected with a mechanical pipette. The chloroform layer was transferred to an HPLC vial, while the polar phase was placed into an Eppendorf tube. Polar phase samples were dried overnight under vacuum concentrator (SpeedVac: Preset 2 until complete solvent evaporation was achieved. Dried pellets of polar phase solutions were resuspended in 50 µL of deuterated phosphate buffer (200 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 200 µM NaN\u003csub\u003e3\u003c/sub\u003e, pH 7.4) with 1 mM internal standard TSP. The reconstituted solutions were sonicated for 1 min in Eppendorf tubes, then centrifuged at 30,000 × g for 30 min to eliminate residual particulate matter. Aliquots of 40 µL from the supernatant were transferred into 5 mm NMR tubes using gel loading pipette tips and placed in a sample rack. The samples were stored at 4°C until NMR spectral acquisition.\u003c/p\u003e\n\u003cp\u003eProton nuclear magnetic resonance (¹H-NMR) spectra were reacquired by a 14.10 Tesla (600 MHz for proton channel) ultra-shielded NMR spectrometer with a 5 mm triple resonance TXI room temperature probe (Avance™ III HD, Bruker BioSpin, Karlsruhe, Germany). For metabolomics analysis, Carr-Purcell-Meiboom-Gill (CPMG) was employed with 256 scans and water suppression used for spectral measurements at 298 K (24.85°C). The recorded free induction decays (FIDs) were Fourier-transformed (FT); phase and baseline corrections were applied. Metabolites annotation and quantification was performed employing commercial software (ChenomX NMR Suite 9.0) and integrated databases (ChenomX and HMDB libraries).\u003c/p\u003e\n\u003cp\u003eStatistical analysis of metabolomics data was performed using MetaboAnalyst 5.0 R-based online analysis tool (http://www.metaboanalyst.ca/). Metabolites were excluded from the analysis if more than 66% of the results for a metabolite were missing. For the remaining metabolites, missing values were replaced with a small value corresponding to 1/5 of the minimum positive value of that metabolite in the original dataset. To account for dilution effects, the data were normalized to a reference sample using probabilistic quotient normalization (PQN) and scaled using Pareto scaling (mean-centred and divided by the square root of its standard deviation of each variable).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTimsTOF LC-MS measurements.\u003c/strong\u003e For comprehensive LC-MS analysis, a chromatography system (Elute Plus, Bruker Daltonics, Bremen, Germany) was coupled to a trapped ion mobility spectrometry time of flight (timsTOF) mass spectrometer (timsTOF Pro 2, Bruker Daltonics, Bremen, Germany) equipped with a vacuum insulated probe heated electrospray ionization (VIP-HESI) source. The instruments were controlled using the Bruker HyStar software and TimsControl acquisition software. Reversed-phase (RP) chromatography was used for the analysis employing an Intensity Solo 2 C18 column (100 mm × 2.1 mm, 2 µm; Bruker Daltonics) was used. The eluent system for mobile phase A was water with 0.1% formic acid, and for mobile phase B, ACN with 0.1% formic acid. The flow rate was set to 0.6 mL/min with a gradient starting at 5% B for the first 2 min, increasing to 60% B at 10 min, 98% B at 11 to 13 min, and returning to 5% B at 13.1 minutes until 15.5 min. The injection volume was 2 µL of the desalted samples.\u003c/p\u003e\n\u003cp\u003eThe MS analysis for non-targeted metabolomics was performed using Parallel Accumulation Serial Fragmentation (PASEF) mode with data-dependent MS/MS acquisition and Trapped Ion Mobility Spectrometry (TIMS) stepping, following the 4D metabolomics standard method in TimsControl software (Bruker Daltonics, Bremen, Germany). The source parameters were set as follows: End Plate Offset at 500 V, Capillary Voltage at 4500 V, Nebulizer Pressure at 2 bar, Dry Gas Temperature at 230°C, Dry Gas Flow at 8.0 L/min, Sheath Gas Temperature at 400°C, and Sheath Gas Flow at 4 L/min. The specific timsTOF Pro 2 parameters included the following: Acquisition Mode in PASEF with TIMS on, Number of PASEF Ramps set to 2, Mass Range from 50–1300 Da, Mobility Range (1/K₀) from 0.10–1.50 V·s/cm², TIMS Ramp Time at 100 ms, Collision RF at 450 Vpp, TOF Transfer Time at 65 µs, Pre Pulse Storage Time at 3 µs, and TIMS Stepping enabled with two steps. The collision energy for fragmentation was set to 20/50 eV. RP chromatography was conducted in both negative and positive ionization mode. Mass and mobility were recalibrated using a 1:3 (v/v) mixture of sodium formate and Agilent’s ESI-L LC/MS Tuning Solution, injected at the start of each run. Additionally, a pooled QC sample (containing same amount from each sample) was used for signal correction during MS analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMS data processing and metabolite annotation\u003c/strong\u003e were performed using MetaboScape 2025 software (Bruker Daltonics, Bremen, Germany). Features have been extracted utilizing the program’s T-ReX-4D™ algorithm with an intensity threshold of 3000 ion counts and minimum 4D peak size of 150 points. Recursive feature extraction was activated with a minimum 4D peak size of 125 points. Metabolites were assigned by matching extracted features against target lists (Bruker HMDB Metabolite Library 2.0, METLIN-CCS Lipid Database, PNNL CCS Metabolites Database, Unified CCS Compendium 2020-03-30, Microbial Metabolites Database Version 1.0) and spectral libraries (Bruker MetaboBASE Personal Library 3.0). The annotations followed MS/MS-based level 2 confidence criteria for metabolite identification, as defined by the Metabolomics Standards Initiative \u003csup\u003e65\u003c/sup\u003e. A match was considered if, in addition to the mass-to-charge ratio (m/z) deviation being \u0026lt; 2.0 ppm, at least one of the following criteria was met: mSigma \u0026lt; 20, MS/MS score \u0026lt; 900, or collision cross-section (CCS) deviation \u0026lt; 1%. The extracted ion chromatograms shapes were also considered. Features with intensities in study samples less than three times those observed in blanks were excluded.\u003c/p\u003e\n\u003cp\u003eEach measurement mode was pre-processed using the MetaboAnalyst web server (Version 6.0, www.metaboanalyst.ca\u003c/a\u003e). Data were uploaded as comma-separated value (.csv) files, with missing values being replaced with 1/5 of the minimum positive value for each feature. Raw data was log transformed to stabilize variance and reduce skewness for each RP mode separately. Features detected by multiple methods were retained from the method with the lowest number of missing values and the highest overall signal intensity.\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eSummary of \u003cem\u003eC. albicans\u003c/em\u003e metabolites associated with virulence and pathogenicity detected in \u003cem\u003eC. albicans\u003c/em\u003e enriched culture with \u003cem\u003eA. fumigatus\u003c/em\u003e secretome. Metabolites are described with their class, metabolite ID (from https://mimedb.org) and their biological function.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003eMetabolite\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eClass\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eMetabolite ID\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eBiological function\u003cbr\u003e\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eDL-Pipecolate\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003emetabolite of lysine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000492\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eBiofilm formation\u003csup\u003e58\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eN6-Acetyl-L-lysine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eN-acyl-alpha amino acids\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000503\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eIdentified in \u003cem\u003eCandida maltose\u003c/em\u003e. The enzyme was strongly induced in cells grown on L-lysine as the sole carbon source\u003csup\u003e29\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003ePyridoxamine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ethe 4-aminomethyl form of vitamin B6\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000195\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eVirulence in \u003cem\u003eC. albicans\u003c/em\u003e and\u0026nbsp;\u003cem\u003eA. fumigatus\u003c/em\u003e\u003csup\u003e66,67\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eD-pantothenic acid\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eVitamine B5\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000069\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eEssential for \u003cem\u003eC. albicans\u003c/em\u003e virulence and yeast survival (\u003cem\u003eA. fumigatus\u003c/em\u003e: virulence and siderophore production)\u003csup\u003e66,67\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eNiacinamide\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eVitamine B3\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000192\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eComponent of the coenzyme NAD in vitamin biosynthesis pathway\u003csup\u003e66,67\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003ePiridoxine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eThe 4-methanol form of vitamin B6\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000081\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eVirulence in \u003cem\u003eC. albicans\u003c/em\u003e and\u0026nbsp;\u003cem\u003eA. fumigatus\u003c/em\u003e\u003csup\u003e66,67\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eTryptophol\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eindolyl alcohol\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0007020\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eFungal quorum sensing molecule\u003csup\u003e48,49\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eTrehalose\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003e1-alpha (disaccharide) sugar\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000177\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eRole in resistance to environmental stress, virulence and survival\u003csup\u003e40,68,69\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eL-acetylcarnitine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eAcyl carnitines\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000501\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eRole in invasion: Affect claudin subtypes (open intestinal tight junctions)\u003csup\u003e70\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eL-phenylalanine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eL-alpha-amino acid\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000048\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eUptake in differenciated hyphae\u003csup\u003e71\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eLysine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eAmino acid\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000059\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eBiofilm related pathway \u003csup\u003e72\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eGlutathione\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eantioxidant\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0029474\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eModulation stress resistance\u003csup\u003e73\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eLactate\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003esecondary alcohols\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0056071\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eCell wall remodelling: thicker cell wall in \u003cem\u003eC. albicans\u003c/em\u003e and reduce immune cell visibility\u003csup\u003e73,74\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cbr\u003e\u003c/div\u003e\u003cbr\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eTable summarizing all the detected \u003cem\u003eAspergillus\u003c/em\u003e mycotoxins by TOF tims LC-MS with their class, metabolite ID (from https:/mimedb.org) and their biological function.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003eMetabolite\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eClass\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eMetabolite ID\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eBiological function\u003cbr\u003e\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eSpirotryprostatin A\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eDiketopiperazine alkaloids\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0001046\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eCytotoxicity, antimicrobial effects, antifungal properties\u003csup\u003e54,55,75,76\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eSphingofungin B\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eLong-chain fatty acids\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0019721\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eSimilar role as fumonisins, antifungal activity\u003csup\u003e53,77\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eSphingofungin D\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePhytoceramides\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0002934\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eAntifungal activity, inhnibit enzyme essential in the biosynthesis of sphingolipids\u003csup\u003e53\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eCyclotryprostatin A\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eBeta carbolines\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0013469\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eInhibitor of the mammalian cell cycle\u003csup\u003e76,78\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eTR-2 verruculogen\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eBeta carbolines, fumitremorgins family\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0012435\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eTremorgenic mycotoxin\u003csup\u003e79,80\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eFumiquinazoline F\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePeptidyl alkaloid\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0019494\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eCytotoxic effect, produced during biofilm formation\u003csup\u003e81–83\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eFumiquinalozine D\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eQuinalozine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eCID 9980845\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eAntibacterial and antifungal (\u003cem\u003eC. albicans\u003c/em\u003e)\u003csup\u003e82–84\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eAculeatsquinone D\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eBenzoquinone derivatives\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0009940\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eCytotoxic, antimicrobial, antifungal\u003csup\u003e85,86\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eAlternariol 4-methyl-10-acetyl ether\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePhenolic compound\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0006882\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eCytotoxic\u003csup\u003e87\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eSperadine B\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eCyclopiazonic acid alkaloids\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0001931\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eCytotoxic activity\u003csup\u003e30,31\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e1-pyrroline-4-hydroxy-2-carboxylate\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePyrrolinecarboxylic acid\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0049981\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eHeat tolerance in fungi\u003csup\u003e88\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e6.7-seco-Agroclavine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eAlkaloid clavines and derivatives\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0013361\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eUnknown\u003csup\u003e89\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eSignificant metabolites found in essential fungal pathway (here in \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eA. fumigatus\u003c/em\u003e) with their class, metabolite ID, biological function and metabolic pathway (from https://mimedb.org).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003eMetabolite\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eClass\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eMetabolite ID\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eBiological function\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eMetabolic Pathway\u003cbr\u003e\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eInosine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePurine mucleoside\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000064\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eSecond messenger\u003csup\u003e41\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eSalvage pathway\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eGuanine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eDerivative of purine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000037\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eHydrolyze bound GTP to GDP\u003csup\u003e41\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eSalvage pathway\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eUMP\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eUracil nucleotide\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0029491\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePrecursor for RNA synthesis\u003csup\u003e33\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePyrimidine pathway\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eUridine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePyrimidine nucleoside\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000094\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eConvert to UMP, crucial for virulence and survival in the host\u003csup\u003e33\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePyrimidine pathway\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eUDP-glucose\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePyrimidine nucleotide sugars\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0032979\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eBeta-glucans precursor cell wall\u003cbr\u003e\u003cem\u003e(C. albicans\u003c/em\u003e and\u0026nbsp;\u003cem\u003eA. fumigatus)\u003c/em\u003e\u003csup\u003e32\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePyrimidine pathway\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eUDP-N-Acetylglucosamine\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eNucleotide sugar\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0029492\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eChitin precursor cell wall (\u003cem\u003eC. albicans\u003c/em\u003e and\u0026nbsp;\u003cem\u003eA. fumigatus)\u003c/em\u003e\u003csup\u003e32\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003ePyrimidine pathway\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eIndoleacetaldehyde\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003e3-alkylindoles\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0032968\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eDerived tryptophan, Convert to Indole-3-acetic acid\u003csup\u003e41,90,91\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eIndole and derivatives pathway\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eIndole-3-carboxaldehyde\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eIndole\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0000271\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eDerived tryptophan, Convert to indole-3-carboxylic acid\u003csup\u003e41\u003c/sup\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eIndole and derivatives pathway\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e(S)-8-amino-7-oxononanoic acid\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMedium-chain fatty acids\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMMDBc0054107\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eSubstrate in biotin pathway https://pubchem.ncbi.nlm.nih.gov/compound/7-Keto-8-Aminopelargonic-Acid)\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eBiotin pathway.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization was done by NB, CT, FD, ST. Data Curation completed by ST, DI and JS. Formal Analysis was done by ST and GB. ST performed Investigation. CT did the research Validation. Supervision was under FD, NB and CT. Writing-Original Draft Preparation was done by ST. Writing-review and Editing was done by NB, CT and FD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Daniele Bucci for their technical support as well as Sisi Deng. Illustrations were in part generated using Biorender.\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Deutsche Forschungsgemeinschaft (German Research Foundation Project 512332979), Bruker Switzerland AG (grant number B23F-1D9C) and the Werner Siemens Foundation. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT reports a research grant by Bruker Switzerland AG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current are available from the corresponding author on reasonable request\u0026nbsp;(lead contact:\u0026nbsp;Christoph Trautwein\u003csup\u003e\u0026nbsp;\u003c/sup\u003e([email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKreitmann, L. \u003cem\u003eet al.\u003c/em\u003e ICU-acquired infections in immunocompromised patients. \u003cem\u003eIntensive Care Med.\u003c/em\u003e 50, 332\u0026ndash;349 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubroto, E., Van Neer, J., Valdes, I. \u0026amp; De Cock, H. 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Prod.\u003c/em\u003e 72, 2069\u0026ndash;2071 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, J. \u003cem\u003eet al.\u003c/em\u003e Biosynthetic Pathways and Functions of Indole-3-Acetic Acid in Microorganisms. \u003cem\u003eMicroorganisms\u003c/em\u003e 11, 2077 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Candida albicans, Aspergillus, fumigatus, fungal interaction, metabolic exchange profiling, H-1-NMR, timsTOF LC-MS","lastPublishedDoi":"10.21203/rs.3.rs-9059703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9059703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eCandida albicans (C. albicans) \u003c/em\u003eand \u003cem\u003eAspergillus fumigatus\u003c/em\u003e (\u003cem\u003eA. fumigatus)\u003c/em\u003e are major opportunistic species, classified by the World Health Organization(WHO) in 2022 as \u003cem\u003ecritical priority pathogen\u003c/em\u003e due to their high morbidity and mortality rates. Despite the prevalence of \u003cem\u003eCandida-Aspergillus \u003c/em\u003ecoinfections in organ transplant recipients and patients with hematologic malignancies, mechanisms underlying their biological interplay remain poorly understood. Using a combinatory metabolomics approach of targeted proton Nuclear Magnetic Resonance spectroscopy and untargeted metabolomics by trapped ion mobility spectrometry time-of-flight mass spectrometry, we investigated metabolic interactions between these pathogens and annotated a total of 176 compounds. We highlighted ten \u003cem\u003eA. fumigatus\u003c/em\u003e mycotoxins, among them, sphingofungin B and D and spirotryprostatin A, decreased in the presence of \u003cem\u003eC. albicans \u003c/em\u003esecretomes, indicating a potential inhibitory effect. Notably, \u003cem\u003eC. albicans\u003c/em\u003e cultures exposed to conidia secretome formed a distinct metabolic cluster. In coculture, an inhibition zone with fragmented \u003cem\u003eA. fumigatus\u003c/em\u003ehyphae was observed, suggesting hyphal damage. Despite this, both species could grow together, highlighting their capacity of cohabitation albeit displaying antagonistic metabolic interactions. The observed inhibition zone and mycotoxin modulation suggest a competitive, yet non-lethal interaction characterized by unique metabolomic chemo-sensing. Importantly, the production of tissue-damaging mycotoxins such as sphingofungin B and D, TR-2 in \u003cem\u003eA. fumigatus\u003c/em\u003e- \u003cem\u003eC. albicans\u003c/em\u003e coinfection may predispose infected sites to polymicrobial invasion, exacerbating disease severity and persistence, impairing treatment efficacy and worsening patient prognosis.\u003c/p\u003e","manuscriptTitle":"Elucidation of the secretory crosstalk between the pathogens Candida albicans and Aspergillus fumigatus via multimodal metabolomics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 15:30:40","doi":"10.21203/rs.3.rs-9059703/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e7d35f2e-c7f2-4f58-a099-dc6b76879c47","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":66638808,"name":"Health sciences/Diseases"},{"id":66638809,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-21T15:30:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 15:30:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9059703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9059703","identity":"rs-9059703","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}