Unveiling Candida albicans intestinal carriage in healthy volunteers: the role of micro- and mycobiota, diet, host genetics and immune response

Abstract

Candida albicans is a commensal yeast present in the gut of most healthy individuals but with highly variable concentrations. However, little is known about the host factors that influence colonization densities. We investigated how microbiota, host lifestyle factors, and genetics could shape C. albicans intestinal carriage in 695 healthy individuals from the Milieu Intérieur cohort. C. albicans intestinal carriage was detected in 82.9% of the subjects using quantitative PCR. Using linear mixed models and multiway-ANOVA, we explored C. albicans intestinal levels with regard to gut microbiota composition and lifestyle factors including diet. By analyzing shotgun metagenomics data and C. albicans qPCR data, we showed that Intestinimonas butyriciproducens was the only gut microbiota species whose relative abundance was negatively correlated with C. albicans concentration. Diet is also linked to C. albicans growth, with eating between meals and a low-sodium diet being associated with higher C. albicans levels. Furthermore, by Genome-Wide Association Study, we identified 26 single nucleotide polymorphisms suggestively associated with C. albicans colonization. In addition, we found that the intestinal levels of C. albicans might influence the host immune response, specifically in response to fungal challenge. We analyzed the transcriptional levels of 546 immune genes and the concentration of 13 cytokines after whole blood stimulation with C. albicans cells and showed positive associations between the extent of C. albicans intestinal levels and NLRP3 expression, as well as secreted IL-2 and CXCL5 concentrations. Taken together, these findings open the way for potential new interventional strategies to curb C. albicans intestinal overgrowth.

Keywords: Candida albicans; GWAS; colonization resistance; host factors; lifestyle factors; metagenomics; microbiota; mycobiota.

Figure 1.

Mycobiota characteristic of healthy subjects. (a). Violin plot of the fungal load observed in the 695 healthy subject fecal samples. (b). Alpha diversity : violin plot of the Shannon Index obtained for 604 healthy subjects’ fecal samples. (c). Beta diversity: Bray-Curtis dissimilarity values between samples donated by different subjects for ITS2 sequencing data obtained for 604 healthy subjects. Values range from 0 to 1, with 0 being the least dissimilar and 1 being the most dissimilar. (d). Barplot of the mean relative abundances of the fungal species that were detected in at least 50% of the 604 studied healthy subjects with a relative abundance above 0.1%. (e). Barplot of the mean relative abundances of the fungal genera that were detected in at least 50% of the 604 studied healthy subjects with a relative abundance above 0.1%. (f) Violin plot of C. albicans DNA levels observed in the 695 healthy subject fecal samples.

Figure 2.

Effect of the age and sex of the subjects on C. albicans carriage. (a). Boxplot of the distribution of C. albicans DNA levels in male and female subjects. (b). Boxplot of the distribution of C. albicans DNA levels depending on the age of the subjects in the subjects colonized by C. albicans. (c) Percentage of subjects colonized by C. albicans (green) and not colonized (white) within each age group. The percentage of colonized and not colonized subjects within each age group are noted in italics *p-value < .05.

Figure 3.

Characterization of bacterial species supernatants and their effect on C. albicans growth. (a). Characterization of the short-chain fatty acids (SCFA) content of each of the bacterial species tested. The SCFA were quantified in the culture supernatants and normalized against the background growth medium control. (b). Boxplot representing the effect of bacterial culture supernatants on the survival of C. albicans, strain SC5314 after 24 h of exposure, relative to the control growth in M2GSC medium (red line, confidence intervals are represented in orange). NGY: C. albicans growth in NGY medium, PBS: C. albicans growth in PBS.

Figure 4.

Comparison of the gut mycobiota of the Milieu intérieur subjects colonized by C. albicans with that of non-colonized Milieu Intérieur subjects. (a). Boxplot of the distribution of fungal Shannon index depending on C. albicans colonization state. (b). Barplot of the mean relative abundances of the fungal species that were detected in at least 50% of the studied healthy subjects with a relative abundance above 0.1%, depending on C. albicans colonization state. (c) Barplot of the mean relative abundances of the non-dominant fungal genera whose mean relative abundance, across subject, was above 0.1%, depending on C. albicans colonization state. Dominant genera are fungal genera detected in at least 50% of the studied healthy subjects with a relative abundance above 0.1%. Non-dominant genera are the remaining fungal genera. *p-value < .05, **p-value < .005.

Figure 5.

Diet and medical factors have a limited impact on C. albicans intestinal carriage and colonization. (a). Boxplot of the variation of C. albicans DNA levels according to the salting habits of the subjects. (b). Boxplot of the variation of C. albicans DNA levels according to the snacking habits of the subjects. (c). Association between C. albicans intestinal carriage and colonization and the mean corpuscular hemoglobin concentration. (left) Boxplot of the distribution of the mean corpuscular hemoglobin concentration depending on C. albicans colonization state. (right) scatterplot of the mean corpuscular hemoglobin concentration relative to intestinal C. albicans DNA levels. The regression line is represented in green, and the interval of confidence in gray. *p-value < .05, **p-value < .005.

Figure 6.

MC3R locus is associated with C. albicans intestinal colonization susceptibility. (a). Manhattan plot of single-nucleotide polymorphisms (SNPs) associated with C. albicans intestinal colonization susceptibility, identified by the genome-wide association study (GWAS) conducted on the 695 subjects of Milieu Intérieur. The x-axis represents the chromosomal position, and the y-axis represents the -log₁₀(p-values) associated with each SNP. The green line represents the suggestive threshold for association (p-value <1.00 × 10⁻⁶). The gray line represents a threshold of 5.00 × 10⁻⁸. (b). Regional association plot for the C. albicans intestinal colonization-associated SNP, rs2870723 (purple diamond). Each dot represents a SNP, the color of the dots corresponds to the linkage disequilibrium of the neighboring SNPs with the top SNP. The x-axis represents the chromosomal position, the left y-axis represents the -log₁₀(p-values) associated with each SNP (dots) and the right y-axis represents the recombination rate (blue line) occurring in each position of the locus.

Figure 7.

Association between rs2870723 genotypes and the levels of C. albicans intestinal carriage. (a) Boxplot of the variation of C. albicans DNA levels according to the rs2870723 genotype of the 695 Milieu Intérieur subjects. (b) Boxplot of the variation of C. albicans DNA levels according to the rs2870723 genotype of the 574 subjects colonized with C. albicans. (c) Percentage of subjects colonized by C. albicans (green) and not colonized (white) according to the rs2870723 genotype of the Milieu Intérieur subjects. The percentage of colonized and not colonized subjects within each genotype are noted in italics. ***p-value < .0005, ns non-significant.

Figure 8.

The extent of C. albicans intestinal carriage was associated with the expression levels of NLRP3 and the concentration of IL-2 and CXCL5 upon ex vivo C. albicans blood stimulation. (a). Residual plots of the association between C. albicans intestinal DNA levels and the expression of NLRP3, and the concentration of IL-2 and CXCL5 upon C. albicans stimulation. Linear model residuals are plotted in relation to the expression of NLRP3 or the concentration of IL-2 and CXCL5. The LOESS line is represented in green and the interval of confidence in gray. (b) Proportion of the expression and concentration variance explained by C. albicans intestinal carriage, age, sex, genetics, and proportions of immune cells for NLRP3, IL-2 and CXCL5, in response to C. albicans blood stimulation.