{"paper_id":"ede35b87-0128-40d7-aabd-a05ce462d31c","body_text":"1 \n \nThe ATO gene family governs Candida albicans  colonisation in the \ndysbiotic gastrointestinal tract \n \nRunning Title: Candida albicans ATO genes promote gut colonisation \n \nRosana Alves\n1, Faezeh Ghasemi1, Wouter Van Genechten 2, Stefanie Wijnants2, \nOdessa Van Goethem 2, Cláudia Barata-Antunes 1, Vitor Fernandes 1, Patrícia \nAtaíde1, Alexandra Gomes-Gonçalves 1, Rudy Vergauwen 2, Qinxi Ma 3, Ricardo \nDuarte1, Isabel Soares-Silva 1, Margarida Casal 1, Alistair J. P. Brown 3, Patrick \nVan Dijck2,4, Sandra Paiva1,# \n \n1Centre of Molecular and Environmental Biology (CBMA), Campus de Gualtar, \nUniversity of Minho, Braga, Portugal \n2Laboratory of Molecular Cell Biology, Kasteelpark Arenberg 31, KU Leuven, \nLeuven, Belgium \n3Medical Research Council Centre for Medical Mycology at the University of \nExeter, Exeter, England, UK \n4KU Leuven One Health Institute, Leuven, Belgium \n#spaiva@bio.uminho.pt \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n2 \n \nABSTRACT \nThe fungal pathogen Candida albicans colonises the human gut where short-chain fatty \nacids (SCFAs) offer sources of carbon. This fungus harbours one of the largest \nmicrobial families of ATO (Acetate Transport Ortholog) genes, which encode putative \nSCFA transport proteins. Here, we generate C. albicans null mutants lacking individual \nor all known putative SCFA transporter genes and compare their phenotypes in vitro and \nin vivo. We show that blocking ATO function in C. albicans  impairs SCFA uptake and \ngrowth, particularly on acetate. The uptake of acetate is largely dependent on a \nfunctional Ato1 (also known as Frp3/Ato3) and it is effectively abolished upon deletion of \nall ATO genes. We further demonstrate that deletion of the entire ATO  gene family, but \nnot inactivation of ATO1 alone, compromises the stable colonisation of C. albicans in the \nmurine gastrointestinal tract following bacterial disruption by broad-spectrum antibiotics. \nOur data suggest that the ATO gene family has expanded and diversified during the \nevolution of C. albicans  to promote the fitness of this fungal commensal during gut \ncolonisation, in part through SCFA utilisation. \n \nIMPORTANCE \nThe human gut is rich in microbial fermentation products such as SCFAs, which serve \nas key nutrients for both bacteria and fungi. C. albicans, a common fungal resident of \nthe gut and a cause of opportunistic infections, carries an unusually large family of ATO \ngenes. This study reveals that this ATO gene family is required for the efficient uptake of \nacetate, the most abundant SCFA in the gut, and for stable colonisation of the gut. \nThese findings uncover a new layer of metabolic adaptation in fungal commensals of \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n3 \n \nhumans and suggest that transporter gene expansion can shape microbial fitness in \nresponse to environmental nutrient signals. \n \nINTRODUCTION \nThe mammalian gastrointestinal (GI) tract is colonised by a complex community of \nmicrobes. Microbial fermentation of dietary fibres generates short-chain fatty acids \n(SCFAs), which are known modulators of host-microbe interactions but also essential \nnutrients that support microbial growth and proliferation in the GI tract (1–3). The \nefficient utilisation of intestinal SCFAs, either as catabolic or anabolic substrates, is \npromoted by specific plasma membrane transport systems.  \nCandida albicans, a human fungal pathogen that has evolved both as a commensal of \nthe GI tract and an opportunistic pathogen, encodes transport proteins from two distinct \ngene families with relevance to SCFA metabolism. The JEN family, which includes the \ncarboxylate transporters JEN1 and JEN2 , is well characterized and known to mediate \ncarboxylate transport (4, 5). In addition, C. albicans  has ten genes from the \nevolutionarily well-conserved acetate uptake transporter (AceTr) family, also known as \nATO genes, which have been proposed to play a role in SCFA uptake (6). This is the \nlargest known ATO family in any microbe (6, 7) and the underlying drivers of the \nexpansion of this family in C. albicans, together with the potential functional \nspecialization of Ato proteins in this species, remain obscure. Indeed, acetate is the \npredominant SCFA in the mammalian GI tract and has long been recognised as a major \ncarbon source for microbes (8, 9).  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n4 \n \nAceTr members are widely distributed across diverse taxa, including bacteria \n(SatP/YaaH) (10–12), archaea (AceP) (7, 13, 14), filamentous fungi (AcpA) (15, 16) and \nyeast (Ady2/Ato1) (6, 7, 17) where they have been reported to play a role in acetate \nuptake. Acetate transporters from bacterial pathogens of humans are the only AceTr \nmembers that have been structurally characterised to date. Two independently solved \ncrystal structures have revealed that bacterial AceTr proteins form a homohexamer \ncomplex (Figure 1A) (11, 12). Each of the six proteins consists of six transmembrane \nsegments with their amino- and carboxy-termini located inside the cell (11, 12) (Figure \n1B). Structural and biophysical studies initially suggested a channel-like complex (11, \n12), but more recent evidence has challenged this idea indicating that acetate \ntranslocation might occur via a carrier mechanism (7, 18). Analyses of alpha-fold (19, \n20) structures for the C. albicans Ato proteins reveal typical architectures analogous to \nthose of bacterial AceTr family members (Figure 1C), suggesting that Ato structural \nfeatures are evolutionarily conserved from prokaryotic to eukaryotic microbes. This \nstructural conservation supports the idea that the primary function of these membrane \nproteins as transporters of acetate, and potentially other SCFAs, has been retained in C. \nalbicans. However, the CaATO9 and CaATO10 genes are most probably nonfunctional. \nWe previously reported that these genes, which encode amino- and carboxy-terminal \nregions of a full length Ato protein (Figure 1C), appear to have evolved from a single \nATO gene that has been split by insertion of a transposable element (6). The Ato9 and \nAto10 proteins consist of only four and two transmembrane segments, respectively \n(Figure 1C).  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n5 \n \nIt is conceivable that an expansion of the ATO gene family might promote rapid \nadaptation of fungal cells to host associated nutrient gradients thereby offering clear \nfitness advantages in the context of human colonisation or infection. Supporting this \nidea, mutations in certain CaATO genes impair the fungus’ ability to neutralize the acidic \nphagolysosome, a trait reported to be linked to hyphal differentiation and survival within \nmacrophages (21, 22). However, it remains unclear whether Ato function is directly \nassociated with virulence (23, 24). Our aim in this study was to investigate the role of the \nATO gene family in SCFA utilisation and assess its contribution on the stable \ncolonisation of the gut. We provide the first direct evidence, beyond structural inference \nfrom bacterial homologs, that Ato proteins mediate acetate transport in C. albicans . \nMoreover, we show that loss of the entire ATO gene family impairs stable colonisation of \nthe mammalian gastrointestinal tract, highlighting the collective and environmentally-\ncontingent role of these transporters in fungal commensalism.  \n \nRESULTS \nInactivation of the ATO gene family in C. albicans \nTo gain further insights into the function of C. albicans  Ato proteins, we generated a \ncollection of single and multiple null mutants using CRISPR-Cas9 systems. Additionally, \nwe constructed mutants lacking the monocarboxylate transporter gene CaJEN1 and/or \nthe dicarboxylate transporter gene CaJEN2 to serve as controls in our assays (4, 5). \nOwing to the diploid nature of C. albicans , the lack of a traditional sexual cycle (25) and \nthe high degree of genomic plasticity, the generation of marker-free homozygous \nknockout strains of multigene families remains technically challenging. Nevertheless, we \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n6 \n \ncreated a set of twelve C. albicans mutants, each lacking a specific JEN or ATO gene, \nas well as strains with multiple JEN and ATO knockouts. Furthermore, we successfully \neliminated all JEN and ATO alleles within a single strain. \n \nATO disruption abolishes acetate utilisation in C. albicans \nWe screened these C. albicans mutants to reveal differential contributions of the 10 Ato \nand 2 Jen proteins to acetate uptake (Figure 2A). As acetate transport via specific \ncarriers follows Michaelis-Menten kinetics (26), using a saturating concentration of 4 mM \nensured that differences in uptake rates between strains can be attributed to genetic \nmodifications rather than suboptimal substrate availability. Wild-type (WT) control cells \ndisplayed 3- to 5-fold higher uptake rates than ato1\n∆ /∆  and multiple Ato-deficient cells \n(Figure 2A, p<0.01). Furthermore, the deletion of JEN transporters in combination with \nATO deletions did not alter the residual acetate uptake capacity (Figure 2A). We then \ncompared the kinetics of radiolabelled acetate transport in WT, ato1∆ /∆ , ato1-10∆ /∆ , \nato1jen1-2∆ /∆  and ato1-10jen1-2∆ /∆  cells for concentrations between 0.5 mM and 4 mM \n(Figure 2B). We confirmed that WT control cells displayed a high capacity  to transport \nacetate (Km 1.59 ± 0.20 mM and Vmax 1.09 ± 0.06 nmol s -1 mg-1, Figure 2B), whereas \nato1∆ /∆  cells showed a significant reduction in acetate uptake ( Km 4.26 ± 1.42 mM and \nVmax 0.51 ± 0.10 nmol s -1 mg-1, Figure 2B). In the absence of ATO  genes ( ato1-10∆ /∆  \nand ato1-10jen1-2∆ /∆  strains), acetate transport was strongly reduced (Figure 2B). The \nobserved residual uptake of radiolabelled acetate in these two strains, which increases \nproportionally with the tested concentrations of acetate, likely represents passive \ndiffusion of the acid into the cell (Figure 2B).  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n7 \n \nThese findings indicate that Ato1 plays an essential role in acetate transport across the \nC. albicans plasma membrane. This hypothesis is supported by Ato1-GFP expression \nand subcellular localization studies, where a clear Ato1-GFP signal was detected at the \nplasma membrane when cells were induced in the presence of acetate (Figure 2C). \nThere was a clear temporal increase in ATO1 expression in response to acetate, in \ncontrast to what is observed in the presence of glucose (Figure 2C). The strong \nfluorescent signal in the plasma membrane observed at 24 hours was also accompanied \nby fluorescence within the vacuole. This pattern is consistent with the well-documented \nintracellular trafficking and recycling of plasma membrane transporters in C. albicans  \nand other fungi (27). Internalisation and delivery to the vacuole often occur after \nprolonged exposure to substrate or as part of turnover and regulation mechanisms (27). \nAdditionally, the growth of ato1\n∆ /∆  cells on acetate as sole carbon source was \ncompletely abolished at 18 ºC but was restored upon ATO1 reintroduction (Figure 2D), \nreinforcing its essential role in acetate utilisation. At this temperature, carboxylic acid \nuptake via passive diffusion is significantly reduced, making growth on carboxylic acids \nentirely dependent on the presence of a functional transporter (7). Despite the potential \nfor genetic redundancy across this family, the presence of other ATO members in \nato1\n∆ /∆  cells did not compensate for the loss of ATO1 in this growth condition. Indeed, \nwe have recently found that loss of ATO1  impairs the expression of other ATOs in the \npresence of acetate (28). Furthermore, ATO1 disruption also impaired cell growth in the \npresence of lactate, similar to the effect observed for the JEN1 disruption (Figure 2D). \nWe then evaluated whether the deletion of additional ATO genes in ato1 ∆ /∆  cells would \nconfer additive growth defects in the presence of acetate or lactate under a more \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n8 \n \nphysiological temperature and growth condition (Figure S1A). Strains carrying additional \nATO gene deletions, particularly ato1-10∆ /∆  and ato1-10jen1-2 ∆ /∆ , did not display any \nexacerbation of the growth defects observed for ato1∆ /∆  cells at 37 ºC, either in \nSynthetic Complete (SC) or Synthetic Minimal (SM) media supplemented with acetate \n(Figure S1A). Moreover, the growth defects observed in ato1∆ /∆  cells were restored \nupon ATO1 reintroduction (Figure S1A). As previously observed at 18 ºC (Figure 2D), \nthe growth of ato1 ∆ /∆  cells was also impaired in the presence of lactate as the sole \ncarbon source at 37 ºC (Figure S1A). However, complete growth abolishment under this \ncondition was only observed upon disruption of JEN1 (Figure S1A).  \n \nATO transporter function promotes gut colonisation during antibiotic-induced \ndysbiosis  \nAltogether, these observations led us to ask whether both ATO  and JEN  families, or \nATO1 alone, influence fungal colonisation in the gastrointestinal tract – a major SCFA-\nrich ecological niche for C. albicans . To test this hypothesis, we performed a series of \nexperiments in a murine model, monitoring the ability of ato and jen mutants to colonise \nthe gut (Figure 3A). The genotypes of all mutants were confirmed by whole genome \nsequencing. The resident microbiota was depleted by treating mice with broad-spectrum \nantibiotics, a prerequisite for C. albicans SC5314 colonisation and a well-documented \nrisk factor for candidiasis (29). We compared the bacterial microbiotas of untreated \n(Figure S2A) versus antibiotic-treated mice (Figures 3B-3D) and the Candida infected \nand uninfected groups by 16S ribosomal RNA gene amplicon sequencing from stool \nsamples (Figures 3B-3D, S2A). Our aim of this amplicon sequencing was to examine \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n9 \n \nthe impact of the antibiotic intervention on the intestinal microbiota, and to reveal any \npotential changes in the gut microbiota associated with colonisation by C. albicans ato \nand jen mutants.  \nThe treated mice displayed decreased alpha-diversity compared with untreated controls \n(p = 0.00016, Figure 3B), which was consistent with the antibiotic-induced depletion of \nbacteria. Similarly, there were significant differences in beta-diversity between untreated \nand antibiotic-treated samples (p = 0.001, Figure 3C). Our data show that the untreated \nmice were mainly colonised by Firmicutes  and Bacteroidota (Figure S2A). Species of \nboth phyla were the main targets of the administered antibiotics, with Bacteroidota \nspecies being partially eliminated (Figure 3D). We also observed a significant increase \nof Actinobacteriota and Proteobacteria species as result of the antibiotic treatment \n(Figure 3D).  \nThese phylogenetic alterations in the intestinal microbiome were consistent with a \ndysbiotic state, often observed after antibiotic treatment (30) and during colonic \ninflammation (31) in both murine models and clinical studies. Candida colonisation did \nnot alter substantially the bacterial microbiota: no significant differences were observed \nbefore and after C. albicans  colonisation (Figures 2D, S2A). Furthermore, our \ncomparisons of groups gavaged with C. albicans WT and knockout  mutants showed \nhigh degrees of similarity in their microbiotas (Figures 3D, S2A).  \nThese analyses were important to ensure that the antibiotic treatment itself did not \nintroduce confounding changes in bacterial community composition, independent of \nfungal colonisation. However, these groups displayed  significant differences in their \nfungal colonisation levels (Figure 3E). C. albicans levels were evaluated by quantifying \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n10 \n \nfungal colony forming units (CFUs) in faeces over the course of seven days. \nSignificantly, none of the tested strains exhibited any fitness defects in vitro in the \npresence of glucose, either in solid (at 18 ºC and 37 ºC, Figures 2D, S1A) or liquid \nmedia (at 30 ºC, Figures S1B-S1C).  \nAs expected (32), control mice that were not treated with antibiotics were not stably \ncolonised by C. albicans  (Figure S2B). In contrast, all tested strains were able to \ncolonise the GI tract of the antibiotic-treated mice (Figure 3E). However, under these \nconditions, WT cells exhibited increased fitness when compared to ato1-10jen1 -2\n∆ /∆  \ncells, with significant differences arising after five days of colonisation ( p < 0.0001, \nFigure 3E). Consistent with this result, ato1-10jen1-2∆ /∆  colonisation levels were also \nreduced, in comparison to WT, when directly assessed in different gut compartments \nsuch as in the small intestine ( p < 0.01, Figure 3F), cecum ( p < 0.01, Figure 3G) and \ncolon ( p < 0.01, Figure 3H). Fungal burdens were consistently lower in the small \nintestine (Figure 3F) than in the cecum (Figure 3G) or colon (Figure 3H), which is in \nagreement with previous observations (33). The significant reduction in the colonisation \nlevels observed for the C. albicans ato1-10jen1-2 mutant compared to both WT and ato1 \nstrains (Figure 3E-H), suggests that ATO gene family members other than ATO1 \npromote C. albicans fitness during gut colonisation.  \nWe also quantified the levels of the key SCFAs acetate, propionate and butyrate in the \ndifferent gut compartments by gas chromatography-mass spectrometry (GS-MS) to \nensure the physiological relevance of our experimental model. The observed levels of \nacetate, propionate and butyrate remained aligned with the classical reported ratio of \n3:1:1 for these acids in the gut (Figures 3I-K) (33). SCFAs levels were reduced in \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n11 \n \nantibiotic-treated mice, consistent with decreased bacterial fermentation under this \ncondition. Nevertheless, these acetate concentrations remained comparable to those \ntested and shown to be dependent upon Ato-mediated uptake in vitro (Figure 2). \n \nDISCUSSION \nOur study demonstrates that the fungal pathogen C. albicans  coordinates the \nassimilation of the key SCFA acetate via the Ato family to facilitate growth and \ncolonisation of the host. Our data are consistent with the idea that AceTr function is \nretained in C. albicans Ato1, with additive effects on acetate utilisation observed as \nmultiple ATO genes are eliminated. While inactivation of ATO1 alone impairs growth on \nacetate, only the deletion of the entire ATO gene family leads to a significant reduction \nin the gut colonisation levels of C. albicans. This suggests functional redundancy and \nhighlights the environmental dependence of ATO gene function. The ability to transport \nacetate confers a metabolic advantage to Candida in the GI tract, where this SCFA is \nabundant and serves as a major available exogenous carbon source. It also enables \nfungal cells to sustain metabolism and survival when thriving in the diverse and complex \nniches of the host. The antibiotic-treated murine model essentially reflects the increased \nrisk of candidiasis that antibiotic treatments pose to humans. Indeed, acetate \nconcentrations in the GI tracts of antibiotic-treated mice (Figures 3I-K) closely mirrored \nthose reported in human faeces following broad-spectrum antibiotic therapy (34), \nthereby validating the physiological relevance of our model.  \nAntibiotic treatment reshapes the gut environment in multiple, interdependent ways \nbeyond simply reducing SCFA pools. The depletion of commensal anaerobes alters \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n12 \n \nniche availability, nutrient flux, and pH , and can also modulate mucosal immune \nresponses and epithelial barrier function (26-28). Such changes may expose novel \nadhesion sites and/or relieve colonisation resistance imposed by bacterial competitors. \nBy probing both single- and multi-gene ato and jen mutants under these complex \nconditions, our study begins to unravel how SCFA uptake pathways interface with host-\nmicrobiota interactions (35). The fact that multiple ATO family members are required for \nrobust colonisation during antibiotic-induced dysbiosis suggests that the functional \ndiversity of this expanded transporter family is critical for fungal persistence in a \ndynamically changing gut ecosystem.  \nIn conclusion, our findings suggest that, during the evolution of C. albicans, the ATO \ngene family has expanded and diversified to promote the fitness of this fungal \ncommensal during gut colonisation, and possibly in other mucosal niches, in part \nthrough the efficient assimilation of SCFAs. We propose that ATOs are significant \nmolecular players in the gastrointestinal colonisation program of C. albicans . Further \nstudies are required to define how individual Ato proteins are regulated, how they \nfunction in concert, and how their activity integrates with broader networks of nutrient \nsensing and host adaptation. \n \nMATERIALS AND METHODS \nMaterials and data availability \nFurther information and requests for resources and reagents should be directed to and \nwill be fulfilled by the corresponding author, Sandra Paiva (spaiva@bio.uminho.pt). All \nunique materials generated in this study are available from the corresponding author \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n13 \n \nwith a completed Materials Transfer Agreement. Any additional information required to \nreanalyse the data reported in this work is also available upon request. \n \nStrains and culture conditions \nAll C. albicans strains used in this study were derived from the wild-type strain SC5314 \nand are listed in Supplementary Table 1. Prototrophic strains were routinely cultured in \nyeast extract-peptone-dextrose (YPD) medium, while nourseothricin-resistant strains \nwere selected on YPD supplemented with 200 µg/mL nourseothricin. For in vitro assays, \ncells were grown at 30 ºC, either in Synthetic Minimal (SM: 0.69% w/v YNB from \nFormedium) or Synthetic Complete (SC: 0.67% w/v YNB from Difco, 0.2% w/v Kaiser \nSC mixture) liquid media supplemented with specific defined carbon sources: glucose \n(0.2% or 2% w/v) or acetate (0.1% v/v, pH 6.0). The culture media pH was adjusted with \na NaOH solution (12M). Solid media was prepared by adding agar (1.5% w/v) to the \nrespective liquid media. \n \nGeneration of CRISPR-cas9 genome edited C. albicans strains \nSingle knockout strains were generated using a transient CRISPR-Cas9 approach, with \nsome modifications. All sgRNA expression cassettes were amplified from pV1093 \nplasmid in three PCR steps using sequentially the following three pairs of primers: \nSNR52/F and SNR52/R_GOI; sgRNA/F_GOI and sgRNA/R; and SNR52/N and \nsgRNA/N, where GOI represents the gene of interest. The Cas9 expression cassette \nwas amplified from pV1093 plasmid with CaCas9/F and CaCas9/R primers. PCR \nreactions for both cassettes were performed according to Min et al (36) without any \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n14 \n \nmodifications. Repair templates were amplified from pV1093 plasmid using \nNAT_GOI_repair/F and NAT_GOI_repair/R primers. The PCR was performed in a total \nreaction volume of 50 \nμ L: pV1093 plasmid (50 ng), 1 μ L forward primer (10 μ M), 1 μ L \nreverse primer (10 μ M), 25 μ L CloneAMP mastermix (Takara), and distilled water up to \n50 μ L. Cycling conditions included an initial denaturation step at 98 °C for 2 min, \nfollowed by 40 cycles of denaturation at 98 °C for 30 s, annealing at 50 °C for 30 s, and \nextension at 72 °C for 1 min, and a final step at 72 °C for 10 min. C. albicans cells were \ntransformed using the classical lithium acetate transformation method (37). The three \ncassettes were co-transformed in a single transformation (1 µg sgRNA cassette, 3 µg \nCas9 cassette and 3 µg repair template). Positive transformants were selected in YPD \nsupplemented with nourseothricin and confirmed by colony PCR with GOI-fwd and GOI-\nrv primers. Multiple-knockout strains and GFP fusions\n (38) were generated using the \nHernday HIS-FLP system(39), with some modifications. For each strain, the specific \ngRNA expression cassette was obtained by cloning-free stitching PCR assembly of \nfragment A (amplified from pADH110 with AHO1096 and AH1098 primers) and fragment \nB (amplified from pADH147 with AHO1097 and Hernday-GOI primers) using AH01237 \nand AH01236 primers. The Cas9 cassette was obtained through digestion of pADH99 \nplasmid with MssI restriction enzyme. Both Cas9 and gRNA cassettes were co-\ntransformed along with a linear DNA fragment (donor DNA) in a single transformation. \nThe 220-bp deletion donors consisted of two 120-bp annealed oligonucleotides with \nhomology to the upstream and downstream flanks of the targeted gene. Positive \ntransformants were selected on YPD supplemented with nourseothricin and confirmed \nby colony PCR. After confirming the intended deletion, the NAT marker along with the \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n15 \n \nCas9 and gRNA expression cassettes were removed from the genome by inducing the \nexpression of the maltose-inducible flipase (FLP) recombinase system on YP 2% \nmaltose liquid medium overnight at 30 ºC. Cells were then streaked on YPD agar for \nsingle-colony isolation and screened on YPD and YPD supplemented with \nnourseothricin to confirm efficient removal of the CRISPR components. All mutant \nstrains were re-checked by colony PCR and validated by sequencing. Selected strains \nfor in vivo  assays were confirmed by whole-genome sequencing. All oligonucleotides \nused in this study are listed in Supplementary Table 2.  \n \nWhole-genome sequencing \nGenomic DNA samples were isolated using DNeasy UltraClean Microbial Kit (Qiagen), \naccording to the manufacturer's recommendations. Whole-genome sequencing was \nperformed at Novogene (UK) using Illumina MiSeqII, with over ~100× coverage. Raw \nsequencing reads were checked for quality using FastQC Software v.0.12.1 \n(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Clean reads were \nassembled into scaffolds using MEGAHIT v.1.2.9 (40). Next, assembly quality was \nassessed using QUAST software v.5.2.0 (41). Gene prediction was performed with \nAUGUSTUS v3.5.0, using C. albicans  SC5314 as the training model to improve \naccuracy. Genes of interest were aligned against each assembled genome using \nBLASTn with default parameters. \n \nPhenotypic assays in liquid and on solid media  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n16 \n \nPhenotypic assays were performed either on solid or liquid media. For evaluation of \ngrowth phenotypes by spot assays, cells were grown in solid YPD (2% w/v) medium. \nCells were then harvested, washed twice in water and for each strain the optical density \nat 640 nm was adjusted to 1. A set of four 1:10 serial dilutions were performed and 3 µL \nof each suspension was spotted into the desired media, either SM or SC. Glucose (0.2% \nw/v) was used as a control carbon source. Cells were inoculated at 18 ºC for 7 days, or \nat 37 ºC for 2 days. For evaluation of growth rates, cells were pre-grown in SC media \nsupplemented with 0.2% glucose (w/v) and then transferred to fresh media with an initial \noptical density of 0.01 at 640 nm. Optical densities were monitored every 2 hours at the \nsame wavelength. The resulting data were processed using GraphPad Prism 9 \nSoftware. Data were log-transformed (base 10), and the exponential phase for each \nindependent experiment was selected. The log of exponential growth equation was used \nfor calculating growth rates. \n \nRadiolabelled Transport assays \nCandida cells were directly grown in SC medium containing acetate (0.1% v/v, pH 6.0), \ncollected at the optical density of 0.5 at 640 nm, washed twice in ice-cold deionized \nwater, and resuspended in water to a final density of 20 – 40 mg (dry weight)/mL. For \neach reaction, a cell suspension of 30 µL was mixed with 60 µL of potassium phosphate \nbuffer (100 mM, pH 6.0). After 2 min of incubation at 30 ºC, each reaction was started by \nthe addition of 10 µL of radiolabelled [1-\n14C]-acetic acid (sodium salt, 55.2 mCi/mmol, \nPerkin Elmer) at the desired concentration for 15 s. Reactions were stopped by dilution \nwith 100 µL ice-cold acetic acid (100 mM, pH 6.0). The reaction mixtures were kept on \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n17 \n \nice, centrifuged at 13 000 rpm, washed with 1 mL ice-cold deionized water and the final \npellet was resuspended in 1 mL of scintillation fluid (Opti-phase HiSafe II). Radioactivity \nwas measured in a Packard Tri-Carb 2200 CA liquid scintillation counter. Each reaction \nwas prepared in duplicate, and each assay was repeated three times. The transport \nkinetics best fitting the experimental initial uptake rates and the kinetic parameters were \ndetermined by a computer-assisted nonlinear regression analysis (GraphPad Software, \nPrism 9).  \n \nFluorescence Microscopy \nC. albicans cells were grown at 30 ºC in SM medium containing glucose (0.2% w/v) until \nmid-exponential phase (OD\n640 nm  = 0.5), washed twice with deionized water and then \ntransferred to fresh SC medium supplemented with acetate (0.1% v/v, pH 6.0) for 6, 8 \nand 24 h. Cells were examined with a Leica Microsystems DM-5000B epifluorescence \nmicroscope with appropriate filter settings. Images were acquired with a Leica \nDCF350FX digital camera and processed with LAS AF Leica Microsystems software. \nPictures are representative of three independent experiments. \n \nMurine model of gastrointestinal colonisation  \nAll animal procedures were performed in accordance with institutional guidelines of KU \nLeuven. The specific protocols for this study were approved by the KU Leuven Ethical \nCommittee (project P010/2020). The laboratory animal usage license number is \nLA1210204. Female C57BL6 mice (aged 8-12 weeks) were obtained from Charles River \nlaboratories. No statistical methods were used to predetermine group size. Each group \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n18 \n \nof mice (n = 4) was assigned at random, housed separately in individual ventilated \nautoclave-sterilized cages, provided with food and water ad libitum  and adapted to \nstandardized environmental conditions (temperature, 23 ± 2 ºC; humidity, 55 ± 10%; 12 \nh light/dark cycles) for at least 1 week before the beginning of each experiment. Four \ngroups of mice were provided with sterile drinking water containing 2 mg/mL \nstreptomycin and 1 mg/m L ampicillin f our days before exposure to C. albicans strains \nand maintained on antibiotic treatment until the end of the experiment. As a control \nexperiment, another four groups of mice did not receive any treatment. Each experiment \n(except control, untreated groups) was repeated twice (n = 8). For experimental \ncolonisation following oral gavage, C. albicans  strains were grown overnight in YPD \nmedium and washed twice in sterile phosphate-buffered saline (PBS). Cell densities \nwere adjusted with PBS and confirmed by flow cytometry-based cell counting and by \nplating (CFUs). Mice were gavaged with 5x10\n7 C. albicans  cells and sacrificed after \nseven days of exposure. Faecal samples were retrieved daily before and after C. \nalbicans colonisation to assay fungal burdens (CFUs) and microbiome composition (16S \nrRNA gene amplicon sequencing). Gut compartments (small intestine, cecum and colon) \nfrom each mouse were harvested in PBS, weighted, homogenized with glass beads \nusing a FastPrep instrument (MP Biomedicals) and then split into samples to determine \nfungal burdens (CFUs) and SCFA concentrations. For CFU counting, both faecal and \ntissue samples were serially diluted tenfold, 50 µL of each dilution plated on \nCHROMagar, and plates grown overnight at 37 ºC. Fungal burdens were expressed as \nCFU per gram of feces or tissue, respectively.  \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n19 \n \n16S rRNA gene amplicon sequencing \nTotal genomic DNA was isolated from stool samples using QIAamp PowerFecal Pro \nDNA Kit (Quiagen) according to manufacturer’s instructions. The DNA concentration and \npurity were determined with NanoDrop 2000 UV-Vis spectrophotometer (Thermo \nScientific). The hypervariable region V3-V4 of the bacterial 16S rRNA gene was \namplified with primer pairs 338F: 5'- ACTCCTACGGGAGGCAGCA-3' and 806R: 5'- \nGGACTACHVGGGTWTCTAAT-3'. Both the forward and reverse 16S primers were \ntailed with sample-specific Illumina index sequences to allow for deep sequencing. The \nPCR was performed in a total reaction volume of 10 \nμ L: 5-50 ng DNA template, 0.3 μ L \nforward primer (10 μ M), 0.3 μ L reverse primer (10 μ M), 5 μ L KOD FX Neo Buffer, 2 μ L \ndNTP (2 mM each), 0.2 μ L KOD FX Neo, and finally ddH 2O up to 20 μ L. Cycling \nconditions included an initial denaturation step at 95 °C for 5 min, followed by 20 cycles \nof denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, and extension at 72 °C for \n40 s, and a final step at 72 °C for 7 min. The amplified products were purified with \nOmega DNA purification kit (Omega) and quantified using Qsep-400 (BiOptic). The \namplicon library was paired-end sequenced (2×250) on an Illumina novaseq6000 \n(Beijing Biomarker Technologies Co). The bioinformatic analysis was performed with the \naid of the BMKCloud (http://www.biocloud.net/). According to quality of single nucleotide, \nraw data was primarily filtered by Trimmomatic v.033 (42). Identification and removal of \nprimer sequences was process by Cutadapt v.1.9.1 (43). PE reads obtained from \nprevious steps were assembled by USEARCH (44) and followed by chimera removal \nusing UCHIME (45). The high-quality reads generated from above steps were used in \nthe following analysis. Sequences with similarity >97% were clustered into the same \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n20 \n \noperational taxonomic unit (OTU) by USEARCH (44), and the OTUs counts less than 2 \nin all samples were filtered. Clean reads then were conducted on feature classification to \noutput an ASVs (amplicon sequence variants) by dada2 (46), and the ASVs counts less \nthan 2 in all samples were filtered. Taxonomy annotation of the OTUs was performed \nbased on the Naive Bayes classifier in QIIME2 (47) using the SILVA database (48) with \na confidence threshold of 70%. Alpha diversity was calculated and displayed by the \nQIIME2 and R software, respectively. Beta diversity was determined to evaluate the \ndegree of similarity of microbial communities from different samples using QIIME. \nPrincipal coordinate analysis (PCoA), heatmaps, UPGMA and nonmetric \nmultidimensional scaling (NMDS) were used to analyse the beta diversity. Furthermore, \nwe employed Linear Discriminant Analysis (LDA) effect size (LEfSe) (49) to test the \nsignificant taxonomic difference among groups. A logarithmic LDA score of 4.0 was set \nas the threshold for discriminative features. To explore the dissimilarities of the \nmicrobiome among different factors, a redundancy analysis (RDA) was performed in R \nusing the package ‘vegan’. \n \nSCFAs extraction and quantification \nSCFAs acids were extracted from gut homogenates colonised by WT C. albicans (small \nintestine, cecum and colon). After homogenization with glass beads using a FastPrep \ninstrument (MP Biomedicals), samples were centrifuged at 15000 rpm for 10 min and \nthe supernatants taken for analysis. For each sample, 2-ethyl butyrate was used as an \ninternal standard at a final concentration of 5 mM. A mixture of SCFAs, 10 mM each \n(lactate, acetate, propionate, formate and butyrate), was used as an external standard. \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n21 \n \nSCFAs were then extracted by the addition of 0.1 mL HCl (37%) and 0.4 mL diethyl \nether to 0.2 mL of each supernatant followed by 1 min of vortex mixing. After \ncentrifugation at 3000 g for 10 min, the ether layer was removed and transferred to a \nseparate capped vial. A further 0.4 mL diethyl ether was added to the aqueous layer and \na second extraction performed. The ether extracts were combined, and 40 µL N-methyl-\nN-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA) added before heating at 80 ºC for 20 \nmin. SCFAs were quantified simultaneously as their tertiary butyldimethylsilyl (t-BDMS) \nderivatives using a gas chromatography-mass spectrometer (Trace 1300 – ISQ QD \nequipped with a TriPlus RSH autosampler and a Restek Rxi-5ms capillary GC column \n30 m x 0.25 mmID). Helium was used as carrier gas with a flow rate of 1.4 mL/min. \nInjection was carried out at 250 ºC in split mode after 1 min and with a ratio of 1:10. The \ntemperature was first held at 50 ºC for 1 min and then allowed to rise to 260 ºC at a rate \nof 50 ºC/min, followed by a second ramp of 2 ºC/min until 325 ºC was reached; that \ntemperature was maintained for 3 min. The mass detector was operated in scan mode \n(50 to 600 atomic mass units), using electron impact ionization (70 eV). The \ntemperatures of the MS transfer line and detector were 325 ºC and 250 ºC, respectively. \nSCFAs were identified by their retention time relative to the internal standard and \nspecific mass spectrometric patterns. Peak areas obtained from the analysis of the \nexternal standard solution, to which internal standard had been added, were used to \ncalculate the relative response factors for each acid with respect to the internal \nstandard. Final concentrations were determined relative to both internal and external \nstandards, and then normalized against tissue weight. \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n22 \n \nStatistics and reproducibility \nAll repeated independent experiments showed similar results. No statistical method was \nused to predetermine sample sizes, and no data were excluded from the analyses. \nStatistical analyses were performed using GraphPad Prism 9 software. All data are \npresented as mean ± sd unless otherwise stated. All details of the statistics, including \nthe specific tests used for each experiment, are provided in the corresponding figure \nlegends. All schematic representations and figures were created or assembled using \nBioRender (https://biorender.com\n). \n \nAcknowledgments \nThis work was supported by the MetaFungal project PTDC/BIA-MIC/5246/2020 \n(https://doi.org/10.54499/PTDC/BIA-MIC/5246/2020), funded by the Portuguese \nFoundation for Science and Technology. Work at CBMA was supported by Contrato \nPrograma UIDB/04050/2020 (https://doi.org/10.54499/UIDB/04050/2020). RA \nacknowledges FEBS for a short-term fellowship at University of Exeter (UK) and FEMS \nfor her stay at KU Leuven (Belgium) in the scope of the FEMS-Jensen Award. FG, PA \nand AG acknowledge FCT for their PhD fellowships (2023.03135.BD; \n2024.03178.BDANA, 2021.08564.BD, respectively). Work at KU Leuven was supported \nby the Research Council (grant #C14/22/075) and the Fund for Scientific Research \nFlanders (FWO grant #G0C0622N).  AJPB was supported by a programme grant from \nthe UK Medical Research Council (MR/M026663/2), a Wellcome Investigator Award \n(224323/Z/21/Z), and by the Medical Research Council Centre for Medical Mycology at \nthe University of Exeter (MR/N006364/2). The funders had no role in study design, data \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n23 \n \ncollection and analysis, decision to publish, or preparation of the manuscript. For the \npurpose of open access, the author has applied a CC BY public copyright licence to any \nAuthor Accepted Manuscript version arising from this submission. \n \nAuthor Contributions \nRA performed the experiments and was responsible for the formal analysis, curation, \nand visualisation of the data. FG, CBA, VF, PA, AG, and QM assisted with the in vitro \nexperiments, while WVG, SW, OVG, and RV assisted with the in vivo experiments. RD \ncontributed to the whole genome sequence analysis. ISS and MC contributed to the \ntransport data analysis. SP, PVD, and AJPB contributed to funding acquisition and were \nmajor contributors in conceptualization, validation and supervision. RA drafted the \noriginal manuscript with additional input from all authors.  \n \nCompeting Interest Statement: The authors declare no competing interests.  \n \nREFERENCES \n1. Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M, Glickman JN, \nGarrett WS. 2013. 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It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n32 \n \nFigure 1. The expanded AceTr transporter family in C. albicans . A. Top and side \nviews of Escherichia coli  SatP crystal structure (PDB ID: 5ZUG), a representative \nprokaryotic member of the acetate uptake transporter (AceTr) family. B. Consensus \nmembrane topology for each protomer (11, 12). C.  Schematic illustration of alpha-fold \npredictions for the 10 Ato membrane proteins in C. albicans. Displayed nomenclature \nfollows new classification (6) (bold) to better reflect and describe their function, while \nmaintaining reasonable consistency with the literature. Standard and systematic names \nare also included.   \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n33 \n \nFigure 2. ATO disruption in C. albicans compromises acetate utilisation. A. Uptake \nof radiolabelled [14C]-acetate (4.0 mM, pH 6.0) into C. albicans WT and knockout strains \ngrown in Synthetic Complete (SC) medium supplemented with acetate (0.1% v/v, pH \n6.0). Error bars indicate mean ± sd of at least 3 independent experiments. Asterisks \nindicate statistical significance between WT and knockout strains, determined using \none-way ANOVA with multiple comparisons tests: **, p < 0.01. B. Uptake rates of \nradiolabelled [ 14C]-acetate, at pH 6.0, as a function of acetate concentration, of cells \ngrown in SC medium supplemented with acetate (0.1% v/v, pH 6.0) as sole carbon \nsource. The kinetic parameters (V max and Km) were determined by a computer-assisted \nnonlinear regression analysis (GraphPad Prism 9.5.0). Error bars indicate mean ± sd of \nn=3 independent experiments. C. Expression and subcellular localization of Ato1-GFP \nthrough time in exponentially growing C. albicans  cells in Synthetic Minimal (SM) \nmedium supplemented with glucose (0.2% w/v) and then transferred to SC medium \nsupplemented with acetate (0.1% v/v, pH 6.0) as sole carbon source for 6, 8 and 24h. D. \nGrowth phenotypes of C. albicans  WT and knockout strains in SM medium \nsupplemented with specific defined carbon sources: glucose (0.2% w/v), acetate (0.1% \nv/v, pH 6.0), lactate (0.1% v/v, pH 5.0). Cells were serially diluted, spotted on solid \nmedia and incubated at 18 ºC for 7 days. Representative data from three independent \nreplicate experiments are shown.  \n \n \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n \n \n34 \n \nFigure 3. C. albicans  ATO proteins allow stable gut occupancy of C. albicans \nunder bacterial dysbiosis. A. Schematic of the experimental design. Mice were \ncolonised with C. albicans strains through oral gavage. Experiments were followed for 7 \ndays post colonisation (dpc). B. Alpha diversity of untreated and antibiotic-treated \ngroups measured by the Simpson index. Data are shown via interquartile ranges with \nthe median as a black horizontal line. C. Beta diversity of untreated and antibiotic-\ntreated groups measured by principal coordinate analysis (PCoA) based on Bray-Curtis \ndistance. D. Relative abundance of taxa (%) in antibiotic-treated mice before (day -1) \nand after C. albicans  colonisation through oral gavage (day 3 and 7). Each bar \nrepresents one independent group of 4 mice. E. Fecal colonisation levels (CFUs/g stool) \nof C. albicans  strains ( n = 8 per strain) in antibiotic-treated mice over time. The \nexperiment was performed twice with similar results. Asterisks reflect comparison \nbetween strains at individual time points using Tukey’s multiple comparisons test: **, p < \n0.01; ***, p < 0.001; ****, p  < 0.0001. F , G, H. C. albicans levels (CFUs/g tissue) in the \nsmall intestine (F), cecum (G), and colon (H) of antibiotic-treated mice (n = 8 per strain) \nat end-point (7 days). Asterisks reflect comparison between strains at individual time \npoints using Tukey’s multiple comparisons test: *, p < 0.05; **, p < 0.01. I , J, K. In vivo \nquantification of SCFAs measured in the small intestine (I ), cecum ( J), and colon \ncolonised by C. albicans (K) by GC-MS. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.  \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 29, 2025. ; https://doi.org/10.1101/2025.05.29.656788doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}