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ABSTRACTOral epithelial cells discriminate between the yeast and hyphal forms of Candida albicansvia the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether β-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of β-glucan and N- and O-mannan mutants, we found that N-mannosylation (via Δoch1and Δpmr1mutants) and O-mannosylation (via Δpmt1and Δmnt1Δmnt2mutants), but not phosphomannan (via a Δmnn4mutant) or β-1,2 mannosylation (via Δbmt1to Δbmt6mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of the N-linked outer chain (corresponding to a Δmnn9mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δmit1mutant) may contribute to epithelial cell damage. β-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicansto induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.

ABSTRACTUnlike other forms of candidiasis, vulvovaginal candidiasis, caused primarily by the fungal pathogen Candida albicans, is a disease of immunocompetent and otherwise healthy women. Despite its prevalence, the fungal factors responsible for initiating symptomatic infection remain poorly understood. One of the hallmarks of vaginal candidiasis is the robust recruitment of neutrophils to the site of infection, which seemingly do not clear the fungus, but rather exacerbate disease symptomatology. Candidalysin, a newly discovered peptide toxin secreted by C. albicanshyphae during invasion, drives epithelial damage, immune activation, and phagocyte attraction. Therefore, we hypothesized that Candidalysin is crucial for vulvovaginal candidiasis immunopathology. Anti-Candidaimmune responses are anatomical-site specific, as effective gastrointestinal, oral, and vaginal immunities are uniquely compartmentalized. Thus, we aimed to identify the immunopathologic role of Candidalysin and downstream signaling events at the vaginal mucosa. Microarray analysis of C. albicans-infected human vaginal epithelium in vitrorevealed signaling pathways involved in epithelial damage responses, barrier repair, and leukocyte activation. Moreover, treatment of A431 vaginal epithelial cells with Candidalysin induced dose-dependent proinflammatory cytokine responses (including interleukin 1α [IL-1α], IL-1β, and IL-8), damage, and activation of c-Fos and mitogen-activated protein kinase (MAPK) signaling, consistent with fungal challenge. Mice intravaginally challenged with C. albicansstrains deficient in Candidalysin exhibited no differences in colonization compared to isogenic controls. However, significant decreases in neutrophil recruitment, damage, and proinflammatory cytokine expression were observed with these strains. Our findings demonstrate that Candidalysin is a key hypha-associated virulence determinant responsible for the immunopathogenesis of C. albicansvaginitis.