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. 2023 Apr 25;14(2):e0010723.
doi: 10.1128/mbio.00107-23. Epub 2023 Mar 1.

A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis-Associated Strains

Arianna Sala  1 , Andrea Ardizzoni  1 , Luca Spaggiari  2 , Nikhil Vaidya  3 , Jane van der Schaaf  3 , Cosmeri Rizzato  4 , Claudio Cermelli  1 , Selene Mogavero  5 , Thomas Krüger  6 , Maximilian Himmel  5 , Olaf Kniemeyer  6 , Axel A Brakhage  6 , Benjamin L King  3   7 , Antonella Lupetti  4 , Manola Comar  8   9 , Francesco de Seta  8   9 , Arianna Tavanti  10 , Elisabetta Blasi  1 , Robert T Wheeler  3   7 , Eva Pericolini  1
Affiliations

A New Phenotype in Candida-Epithelial Cell Interaction Distinguishes Colonization- versus Vulvovaginal Candidiasis-Associated Strains

Arianna Sala et al. mBio. .

Abstract

Vulvovaginal candidiasis (VVC) affects nearly 3/4 of women during their lifetime, and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a switch from a commensal to pathogenic state, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e., multilocus sequence type [MLST] profile, rate of growth, and filamentation), but they show strikingly different behaviors during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal proliferation and shedding of fungi and epithelial cells. Transcriptome sequencing (RNA-seq) analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis, and type I interferon pathways; the latter has been implicated in damage protection. Strikingly, inhibition of type I interferon signaling selectively increases fungal shedding of strains in the colonizing cohort, suggesting that increased shedding correlates with lower interferon pathway activation. These data suggest that VVC strains may intrinsically have enhanced pathogenic potential via differential elicitation of epithelial responses, including the type I interferon pathway. Therefore, it may eventually be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis. IMPORTANCE Despite a high incidence of VVC, we still have a poor understanding of this female-specific disease whose negative impact on women's quality of life has become a public health issue. It is not yet possible to determine by genotype or laboratory phenotype if a given Candida albicans strain is more or less likely to cause VVC. Here, we show that Candida strains causing VVC induce more fungal shedding from epithelial cells than strains from healthy women. This effect is also accompanied by increased epithelial cell detachment and differential activation of the type I interferon pathway. These distinguishing phenotypes suggest it may be possible to evaluate the VVC pathogenic potential of fungal isolates. This would permit more _targeted antifungal treatments to spare commensals and could allow for displacement of pathogenic strains with nonpathogenic colonizers. We expect these new assays to provide a more _targeted tool for identifying fungal virulence factors and epithelial responses that control fungal vaginitis.

Keywords: Candida albicans; epithelial cells; host-pathogen interactions; interferons; vulvovaginal candidiasis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Unrooted neighbor-joining tree showing p-distance for C. albicans clinical isolates typed by MLST, using SC5314 as reference strain. The number for each cluster node indicates the bootstrap value. Bootstrapping was set at 500.
FIG 2
FIG 2
Sequence comparisons of candidalysin sequences at the protein and DNA levels from vaginal isolates. (A) Cladogram of aligned protein sequences with relative distances by nearest-neighbor joining. The 50711 sequence groups away from all the other sequences and matches with the sequence from the 529L clinical isolate. (B) Peptide alignment. Highlighted in green are positions with variability even within the SC5314-type allele group. Conservation among the 22 individual alleles is indicated as fully conserved (*), conservative (:), semiconservative (.), or nonconservative (blank) below the alignment. Protein alleles marked with a # to the left indicate those where candidalysin was detected in vitro by proteomics. (C) Cladogram of aligned nucleotide sequences with relative distances by nearest-neighbor joining; (D) nucleotide alignment. Highlighted in green are positions with variability even within the SC5314-type allele group. Fully conserved positions are denoted with asterisks below the alignment. Alleles are indicated by strain number and then -A and -B to denote variable alleles in a single strain. “Col” indicates a strain from an asymptomatic individual, and “VVC” denotes a strain from a VVC patient.
FIG 3
FIG 3
C. albicans hyphal fragment production. Data in the graphs show the mean percentage ± standard deviation (SD) of hyphal fragments produced by VVC and colonizing C. albicans strains after 24 h of culture in RPMI 1640 (protocol i) (A), in RPMI or RPMI plus 10% FCS in the presence or absence of Lactobacillus rhamnosus (L.r.) (protocol ii) (B), or during infection of a monolayer of A431 cells (protocol iii) (C). Statistical analysis was performed according to Student's t test (A and C) and Kruskal-Wallis test, followed by Dunn’s multiple-comparison test or one-way ANOVA, followed by Tukey’s multiple-comparison test (left and right graphs of panel B, respectively). Panels D and E show representative images of hyphal segments from the 01887 (D) and 14314 (E) strains from experimental protocol ii, grown in sgRPMI. Arrowheads indicate the septa separating one hyphal segment from another. The data come from at least 3 biological replicates.
FIG 4
FIG 4
Vaginal epithelial cell damage and IL-1β production. (A and B) Vaginal cell damage (LDH release) after 24 h of infection with VVC and colonizing C. albicans strains or by the reference strain SC5314 at an MOI of 1:1 (A) or 1:5 (B). The blue dots highlighted in panel A indicate results from an additional group of 9 isolates the provenance of which had been previously reported (see reference 44). (A) Mean ± SD of each cohort; (B) median of each cohort with 95% confidence interval (CI). Each data point represents the average from three biological replicates. (C) IL-1β (picograms per milliliter) released by vaginal cells after 24 h of infection with VVC and colonizing C. albicans strains or by the reference strain SC5314 at an MOI of 1:5; (C) mean ± SD of each cohort. Each data point represents the average from 4 biological replicate experiments. Statistical analysis was performed according to unpaired Student's t test (A; MOI of 1:1) and Mann-Whitney test (B and C; MOI of 1:5), according to data normality. P values of >0.05 were considered not significant and are shown above the data.
FIG 5
FIG 5
C. albicans shedding from vaginal epithelium. (A) Shedding of epithelial and fungal cells was measured by either the shedding protocol (A, upper) or the exfoliation protocol (A, lower). Epithelial or fungal cells could be attached to the substrate, loosely attached to the substrate, or shed in suspension. Each protocol measures different fractions of the total challenge, as indicated by the workflow. The shedding protocol measures shed cells in suspension (top panels), whereas the exfoliation protocol measures shed cells bound to exfoliated cells and/or loosely associated with the epithelium (bottom panels). (B to G) Data in the graphs show the mean ± SD CFU from 3 different experiments after 24 h of infection of the vaginal epithelial monolayer with the indicated C. albicans strains at an MOI of 1:1. (B to D) Shedding protocol. (B) Overall C. albicans CFU for each infection (all cells); (C) C. albicans CFU shed in the supernatants (shed cells in suspension), and (D) C. albicans CFU attached to or loosely associated with vaginal epithelium (exfoliated, adherent, and loosely adherent cells). (E to G) Exfoliation protocol. (E) C. albicans CFU shed in supernatant in association with shed epithelial cells (exfoliated and loosely adherent cells). In panel E, the black dots represent the standard 16 clinical strains used throughout the study, blue dots indicate an additional group of 9 isolates tested (see reference 44), and magenta dots indicate a third group of 10 isolates from women with RVVC or healthy colonized women obtained from IRCSS Burlo Garofolo Trieste, Italy. Statistical analysis was performed according to the Mann-Whitney test. P values of <0.05 (*) and <0.001 (***) were considered significant. P values of >0.05 are not considered significant and are shown above the data. Panels F and G show representative images of C. albicans shedding of the VVC strain CA01887 (F) and the colonizing strain CA14314 (G). Exfoliated and loosely adherent cells are shown, including epithelial cells with punctate CFDA in the cytoplasm (marked by asterisks) and Candida cells (blue with bright dots of CFDA staining). Scale bar = 20 μm.
FIG 6
FIG 6
CA01887 and CA14314 differentially activate type I interferon, integrin, and ferroptosis pathways. A431 vaginal epithelial cells were challenged with either CA01887 or CA14314 for 24 h. Differential RNA-seq was performed on two biological replicates of mock-infected versus infected challenges and analyzed with DEseq2 and Ingenuity Pathway Analysis. (A to C) Differentially regulated genes from three pathways with divergent responses upon C. albicans challenge are shown with statistically significant upregulation (yellow), significant downregulation (blue), or no significant change (white). The range for each is shown below (values are the log2 ratio of fungal challenge to mock challenge). The cutoff for significant changes was an adjusted P value (Padj) of <0.01. (D to F) Summary of IFNAR inhibition of shedding for several VVC and colonizing strains. Shown is the mean percentage of change ± SD in CFU from at least 3 different experiments after 24 h of infection of the vaginal epithelial monolayer with VVC or colonizing strains in the presence or absence of neutralizing IFNAR antibody (Ab). All experiments used the shedding protocol. (D) C. albicans CFU shed in the supernatants (cells in suspension); (E) C. albicans CFU attached to vaginal epithelium (exfoliated, adherent, and loosely adherent cells); (F) overall C. albicans CFU for each infection (all cells). Red dots indicate the strains CA01887 and CA14314, which were used in the original RNA-seq experiments. The statistical comparisons between VVC and colonizing strains were performed according to the unpaired Student's t test. P values of <0.05 (*) were considered significant. n.s., not significant (P > 0.05).
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