Processing of Candida albicans Ece1p is critical for Candidalysin maturation and fungal virulence

Candida albicans is an opportunistic fungal pathogen responsible for superficial and life threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete Candidalysin, a 31 amino acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein-1 (AP-1) transcription factor c-Fos (via p38- MAPK) and the MAPK phosphatase MKP1 (via ERK1/2- MAPK), which trigger and regulate pro44
inflammatory cytokine responses, respectively. The Candidalysin toxin resides as a discrete cryptic sequence within a larger 271 amino acid parental pre-pro-protein, Ece1p. Here we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two47 step post-translational processing of Ece1p to produce Candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature Candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature Candidalysin. C. albicans strains harbouring mutations of Arg61 and/or Arg93 did not secrete Candidalysin, were unable to induce epithelial damage and inflammatory responses in vitro, and showed attenuated virulence in vivo in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of C. albicans Ece1p by kexin-like proteinases as crucial steps required for Candidalysin production and fungal pathogenicity.