Essential requirements for Candida albicans mediated damage of epithelial cells

Selene Mogavero1, Stefanie Allert1, Annika König1, Lydia Kasper1, Jonathan Richardson2, Thomas Krüger3, Olaf Kniemeyer3, Julian Naglik2 and Bernhard Hube1,4,5

1Department of Microbial Pathogenicity Mechanisms, Hans Knöll Institute, Jena, Germany; 2Mucosal and Salivary Biology Division, Dental Institute, King’s College London, UK; 3Molecular and Applied Microbiology, Hans Knöll Institute, Jena, Germany; 4Friedrich Schiller University, Jena, Germany; 5Integrated Research and Treatment Center, Center for Sepsis Control and Care, Jena, Germany.

Objectives
Candida 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.

Methods
To 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.

Results
We 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. 

Conclusions
C. 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.


8 / Essential requirements for Candida albicans mediated damage of epithelial cells