Comparative Analysis of the Capacity of the Candida Species To Elicit Vaginal Immunopathology

Abstract

The human fungal pathogen Candida albicans is the major etiological agent of vulvovaginal candidiasis (VVC). Despite this fact, other non-albicans Candida (NAC) species have frequently been reported, as well. Despite their presence in the vaginal environment, little is known about their capacities to elicit immune responses classically associated with C. albicans-mediated immunopathology, including neutrophil recruitment and proinflammatory cytokine signaling. Therefore, using a combination of in vitro and in vivo approaches, we undertook a comparative analysis to determine whether a representative panel of NAC species could colonize, induce immunopathological markers, or cause damage at the vaginal mucosa. Using a murine model of VVC, C. albicans was found to induce robust immunopathology (neutrophils and interleukin 1β [IL-1β]) and elicit mucosal damage. However, all the NAC species tested (including C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. glabrata, and C. auris) induced significantly less damage and neutrophil recruitment than C. albicans, despite achieving similar early colonization levels. These results largely correlated with a notable lack of ability by the NAC species (including C. dubliniensis and C. tropicalis) to form hyphae both in vitro and in vivo Furthermore, both C. dubliniensis and C. tropicalis induced significantly less expression of the ECE1 gene encoding candidalysin, a key fungal virulence determinant driving VVC immunopathology. In order to determine the relative capacities of these species to elicit inflammasome-dependent IL-1β release, both wild-type and NLRP3^-/- THP-1 cells were challenged in vitro While most species tested elicited only modest amounts of IL-1β, challenge with C. albicans led to significantly elevated levels that were largely NLRP3 dependent. Collectively, our findings demonstrate that although NAC species are increasingly reported as causative agents of VVC, C. albicans appears to be exceedingly vaginopathogenic, exhibiting robust immunopathology, hypha formation, and candidalysin expression. Thus, this study provides mechanistic insight into why C. albicans is overwhelmingly the major pathogen reported during VVC.

Keywords: Candida; NAC species; VVC; immunopathogenesis; inflammasome; vaginitis; vulvovaginal.

FIG 1

NAC species fail to elicit robust inflammation or mucosal damage at the vaginal mucosa. Groups of estrogen-treated C57BL/6 mice (n = 8) were intravaginally challenged with PBS, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. glabrata, or C. auris. Vaginal lavage fluid was assessed longitudinally at day 3 and day 7 for fungal burden (the horizontal lines indicate medians) by microbiological plating (A and B), for PMNs (means plus SEM) by microscopy (C and D), for the damage biomarker LDH (means plus SEM) by enzymatic assay (E and F), or for IL-1β (means plus SEM) by ELISA (G and H). Experiments in all the inoculation groups were performed in duplicate, and the data were combined. The data were tested for normality using the Shapiro-Wilks test. Statistical significance was calculated using one-way ANOVA and the Kruskal-Wallis posttest (nonnormally distributed data) or Dunnett’s posttest (normally distributed data). *, P  < 0.05.

FIG 2

NAC species largely fail to robustly form hyphae or recruit neutrophils in vivo. Vaginal lavage fluids (10 μl) from day 3 p.i. were smeared onto glass slides, fixed, and stained by the Papanicolaou technique. Images of five nonadjacent fields were captured by standard light microscopy, and a representative of each is depicted for C. albicans (A), C. dubliniensis (B), C. tropicalis (C), C. parapsilosis (D), C. krusei (E), C. glabrata (F), C. auris (G), and mock-challenged mice (H). The green arrows indicate fungi, and the yellow arrows indicate neutrophils. Scale bars, 20 μm.

FIG 3

NAC species fail to robustly activate the NLRP3 inflammasome. C. albicans and NAC species (C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. glabrata, and C. auris) were added to differentiated WT and NLRP3^−/− THP-1 cells at an MOI of 5:1 for 4 h. Cells were also challenged with LPS plus ATP as a positive control for inflammasome activation. Cell-free culture supernatants were analyzed for IL-1β release. The solid bars represent WT cells, and the open bars depict NLRP3^−/− cells. The numbers of technical replicates (n = 4) per experiment were averaged. The data are depicted as the average of 3 independent biological repeats (means plus SEM). Statistical significance was calculated using one-way ANOVA and Dunnett’s posttest. Comparison between WT and NLRP3^−/− cells, *, P < 0.05, and **, P < 0.01; comparison between species, #, P < 0.05.

FIG 4

C. dubliniensis and C. tropicalis fail to strongly upregulate ECE1 expression in vitro or in vivo. (A) Overnight YPD cultures of C. albicans, C. dubliniensis, and C. tropicalis were transferred to either fresh YPD or RPMI medium for 4 h. RNA was extracted, normalized, and reverse transcribed, and the expression of ECE1 and the housekeeping gene ACT1 was monitored. The normalized fold expression of ECE1 (mean plus SEM) was calculated from the average independent biological repeats (n = 3) using the ΔΔC_T method. Statistical significance was calculated using one-way ANOVA and Dunnett’s posttest. *, P  < 0.05; ***, P < 0.001. (B) C. albicans, C. dubliniensis, and C. tropicalis were intravaginally inoculated in estrogen-treated mice as described in the text. At day 3 p.i., the mice underwent two rounds of vaginal lavage with PBS to recover fungal cells, and RNA was immediately prepared as described above. The normalized fold expression of ECE1 (mean plus SEM) was calculated from the average of independently inoculated animals (n = 4) and compared to ACT1 expression using the ΔC_T method. n.d., not detected.