A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis

Abstract

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion

“…C. albicans isolates HUN96 and 101 secreted detectable levels of candidalysin and induced PM blebs ( Figure S1 D). In contrast, we failed to detect candidalysin and PM blebs in pockets formed by isolate 529L ( Figure S1 D); this strain expresses a variant candidalysin that is secreted at a reduced rate ( Liu et al., 2021 ). We attribute the failure to form blebs to the reduced secretion, rather than to structural differences between the candidalysins, because ab initio structure predictions for the candidalysin peptides secreted by SC5314 and 529L showed only minor differences that do not markedly affect their hydrophobicity, charge, or polarity ( Figures S1 E vs. S1F).…”

“…One isolate tested, 529L, formed hyphae but did not secrete detectable candidalysin nor did it induce membrane blebs. Notably, the 529L isolate encodes a variant of Ece1, which is processed less efficiently ( Liu et al., 2021 ). Consequently, less mature candidalysin is delivered into the invasion pocket ( Figure S1 F), and damage levels caused by invading 529L hyphae remain low.…”

“…C-QUARK ( https://zhanggroup.org/C-QUARK/ ) was used to generate ab initio structure predictions for the candidalysin peptides sequences available for C. albicans isolates SC5314 ( Moyes et al., 2016 ) and 529L ( Liu et al., 2021 ). Generated PDB files were then modeled and annotated in PyMOL (Schrödinger).…”

Candida albicans commensalism in the oral mucosa is favoured by limited virulence and metabolic adaptation

“…albicans depends not only on strong Ece1 expression but also coordinated delivery of fully processed and secreted Candidalysin in invasion pocket created by invasive hyphae [ 37 ]. Of note, we expressed the entire ECE1 gene originating from SC5314 under the strong TDH3 promoter, including the sequences flanking peptide 3 for optimal Candidalysin processing, to exclude suboptimal processing of Ece1p as recently reported [ 38 ].…”

Candidalysins Are a New Family of Cytolytic Fungal Peptide Toxins

“…Candidalysin is critical for C. albicans mucosal infections and is produced by targeted proteolytic processing of the hypha-associated protein Ece1p by kexin proteases at conserved lysine-arginine recognition sites ( 6 , 7 ). Following secretion, candidalysin destabilizes the plasma membrane of epithelial cells ( 3 , 8 ), triggers cellular stress resulting in necrotic cell death ( 9 ), and facilitates fungal translocation across gastrointestinal epithelial cells ( 10 ).…”