Search

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–mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]–MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational 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 harboring 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., IMPORTANCE Candida albicans is an opportunistic fungal pathogen that causes mucosal infection in millions of individuals worldwide. Successful infection requires the secretion of candidalysin, the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is derived from its parent protein Ece1p. Here, we identify two key amino acids within Ece1p vital for processing and production of candidalysin. Mutations of these residues render C. albicans incapable of causing epithelial damage and markedly reduce mucosal infection in vivo. Importantly, candidalysin production requires two individual enzymatic events. The first involves processing of Ece1p by Kex2p, yielding immature candidalysin, which is then further processed by Kex1p to produce the mature toxin. These observations identify important steps for C. albicans pathogenicity at mucosal surfaces.

Essential requirements for Candida albicans mediated damage of epithelial cellsSelene Mogavero1, Stefanie Allert1, Annika Ku00f6nig1, Lydia Kasper1, Jonathan Richardson2, Thomas Kru00fcger3, Olaf Kniemeyer3, Julian Naglik2 and Bernhard Hube1,4,51Department of Microbial Pathogenicity Mechanisms, Hans Knu00f6ll Institute, Jena, Germany; 2Mucosal and Salivary Biology Division, Dental Institute, Kingu2019s College London, UK; 3Molecular and Applied Microbiology, Hans Knu00f6ll Institute, Jena, Germany; 4Friedrich Schiller University, Jena, Germany; 5Integrated Research and Treatment Center, Center for Sepsis Control and Care, Jena, Germany.ObjectivesCandida albicans, an important opportunistic fungal pathogen of men, is able to transition from the yeast morphology, usually associated with the commensal life style in the gut, to a filamentous hyphal state. This filamentous morphology has long been associated with the invasive and destructive stage of C. albicans, and is accompanied by the expression of several hyphae-associated genes. Among these, ECE1 is one of the most highly transcribed during hyphal growth. The role of ECE1 during mucosal infection has been elucidated: it encodes a polyprotein that is processed by Golgi-associated Kex proteases into several peptides, which are then secreted in the extracellular space. One of these is a peptide toxin, Candidalysin, which is able to damage human host cells.Of all Ece1 peptides, Candidalysin seems to be the first to be secreted and the most abundant in supernatants. Here we set to elucidate the dynamics of expression, processing and secretion of the Ece1 peptides, as important steps preceding delivery of Candidalysin to induce pore-forming mediated damage of epithelial cells.MethodsTo understand the dynamics of expression, processing and secretion, we performed a systematic screening of several mutants defective in damage potential and/or hyphal formation and/or that are linked to Ece1 processing. The mutants were screened for the following features: quantification of ECE1 transcripts by RT-qPCR, ability to cause damage of oral epithelial cells using cytotoxicity assays, ability to form hyphae by measuring hyphal length, and composition of the secretome by LC-MS analysis.ResultsWe identified mutants with reduced or no ECE1 expression, with lower or no ability to form hyphae, secreting less or no Ece1-derived peptides and with reduced or no damaging potential. Low damage, compared to the wild-type, correlated with low ECE1 expression and/or shorter hyphae and/or mutated ECE1 sequences, especially around the Candidalysin sequence. Of note, mutants, where specific KR cleavage sites recognized by Kex2 had been modified to KA, lost their ability to properly release Candidalysin and to damage host cells. ConclusionsC. albicans uses its hyphal morphology to cause damage to host cells, and this is mediated by the Candidalysin peptide toxin. It is clear that, whenever at least one of the two requirements fails, or does not reach a specific threshold, the fungus is handicapped and unable to cause damage. This goes both ways, that is, whenever a mutant is unable to cause damage, the reason will likely be an indirect effect of reduced ECE1 expression or reduced delivery of Candidalysin to the host cells, either because hyphae are shorter, or because the processing or secretion is somehow impaired. In agreement with this conclusion, an optimal ratio of hyphal invasion, hyphal length and Candidalysin secretion is required for full damage.

The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80D mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80D mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.