Passage through the mammalian gut triggers a phenotypic switch that promotes Candida albicans commensalism

Abstract

Among ∼5,000,000 fungal species, C. albicans is exceptional in its lifelong association with humans, either within the gastrointestinal microbiome or as an invasive pathogen. Opportunistic infections are generally ascribed to defective host immunity but may require specific microbial programs. Here we report that exposure of C. albicans to the mammalian gut triggers a developmental switch, driven by the Wor1 transcription factor, to a commensal cell type. Wor1 expression was previously observed only in rare genetic backgrounds, where it controls a white-opaque switch in mating. We show that passage of wild-type cells through the mouse gastrointestinal tract triggers WOR1 expression and a novel phenotypic switch. The resulting GUT (gastrointestinally induced transition) cells differ morphologically and functionally from previously defined cell types, including opaque cells, and express a transcriptome that is optimized for the digestive tract. The white-GUT switch illuminates how a microorganism can use distinct genetic programs to transition between commensalism and invasive pathogenesis.

Figure 1. EFG1 inhibits and WOR1 promotes C. albicans fitness in the commensal milieu

A) Competition experiment between wild-type (WT, SN250) and efg1ΔΔ mutant (SN1011) strains in 4 mice. Relative abundance was determined by qPCR, using strain-specific primers. Log₂(R/I) represents the log ratio of the abundance of each strain after recovery from the feces of a given animal (R) compared to the level in the infecting inoculum (I). * indicates p<0.001 by the t-test (two-tailed, unpaired samples). Similar results were obtained when the efg1ΔΔ mutant was competed in a pool of 48 strains, as described in the text (Pande and Noble, unpublished). B) Transcriptional regulation of the white-to-opaque switch. C) Competition experiment between WT (SN250) and wor1ΔΔ (SN881) in 3 mice, as above. * p<0.05. Similar results were obtained in tests of two additional wor1ΔΔ strains, shown in Supplementary Figure 1, A–B. D) Comparison of WOR1 expression in MTLa/α cells grown in the commensal model or in vitro. The WOR1_promoter-FLP strain (SN1020) was propagated for 3 days in the murine model or for 8 generations in liquid culture medium, followed by 5-FOA selection and PCR of 5-FOA^R colonies to verify Flp-mediated deletion URA3. Mean percentages of cells meeting these criteria are plotted on the y-axis. Error bars reflect the standard deviation among 6 (in vivo) or 4 (in vitro) biological replicates.

Figure 2. Wor1 promotes a white-to-GUT transition that confers enhanced fitness in the mammalian gastrointestinal tract

A) Competition between wild-type (SN425) and WOR1^OE (SN928) MTLa/α strains in the murine commensal model (n = 10 animals). The t-test (two-tailed, unpaired samples) was used to determine significance. *p<0.05, **p<0.001, ***p<0.0001, n.s. nonsignificant. A replicate of this experiment yielded similar results (Pande and Noble, unpublished). B) Appearance of distinct colony morphologies after recovery from the mouse. W signifies colonies with typical white morphology. Colonies with GUT morphology are unmarked. C) and D) Light microscopic appearance of cells recovered from white and GUT colonies, respectively. E) Analysis of colonies recovered from the WT vs. WOR1^OE commensal competition experiment. White (W) vs. GUT (G) phenotypes were visually assessed, followed by determination of strain identity (WT vs. WOR1^OE) by colony PCR.

Figure 3. GUT cells are distinct from previously identified opaque cells

A) Scanning electron micrographs showing “pimples” on the surface of opaque (SN967) but not GUT (SN1045) cells. B) Opaque but not GUT cells form mating filaments in response to mating pheromone (αF). Arrowheads indicate mating projections. All images were obtained at the same magnification. C) Opaque cells (SN967) are significantly outcompeted by wild type (SN425) in the murine commensal model (n = 7 animals). ** p<0.005, *** p<0.001 by the t-test. A replicate of this experiment yielded similar results (Pande and Noble, unpublished).

Figure 4. GUT and opaque cells exhibit overlapping but distinct patterns of transcriptional expression

RNAs from WOR1^OE MTLa/α GUT cells (SN1045, n = 4 biological replicates), MTLa opaque cells (SN967, n = 2 biological replicates), and isogenic white controls (n = 2–4 biological replicates) were profiled using custom Agilent C. albicans ORF microarrays and a pooled reference. Significant changes in gene expression were defined as ones with BAGEL p-values <0.05, after implementation of a Bonferroni correction for comparison of multiple variables. A) Venn diagram of genes that are significantly upregulated or downregulated in both opaque and GUT cells, relative to white cells. B) Genes that are differentially expressed by GUT vs. opaque cells.