Genetic interrogation of virulence in Candida albicans; the major fungal pathogen of humans

Candida albicans is an opportunistic fungal pathogen that causes superficial and life-threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast (commensal) to invasive filamentous hyphae (pathogen) that damage host tissue. Current research demonstrates that the invasive hyphae of C. albicans use multiple virulence factors to cause disease in the human host. Among these, adherence to the mucosal surface, production of a cytolytic toxin and enzymatic degradation of host proteins are major pathogenic attributes that facilitate disease progression.

The major hyphal adhesin of C. albicans is Als3p, which mediates attachment to host tissue through its interaction with the epidermal growth factor receptor (EGFR). Als3p is also a major fungal invasin that drives receptor-induced endocytosis of hyphae into host tissue. The hyphae of C. albicans can damage a range of host cell types by secreting candidalysin, a peptide toxin encoded by the gene ECE1, while secreted aspartyl proteinases (SAPs), particularly Sap2p are involved in the degradation of host proteins.

While studies demonstrate that C. albicans mutants unable to produce either Als3p, candidalysin or Sap2p exhibit diminished pathogenicity, we hypothesise that removal of all three factors may render C. albicans avirulent during both mucosal and systemic infection.

This proposal will investigate the combined role of C. albicans Als3p, candidalysin and Sap2p during fungal infection of human epithelial cells.

The goals of this PhD project are to:
1. Construct an als3/ece1/sap2 triple null mutant in C. albicans.
2. Characterise the C. albicans mutant phenotype using human oral epithelial cells in vitro.
3. Determine the pathogenicity of the C. albicans triple mutant using human in vitro and murine in vivo models.

The als3/ece1/sap2 triple null mutant will be created using established CRISPR technology optimised for use in C. albicans and validated by whole genome sequencing. Successful deletion of each target gene will be confirmed using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Characterisation of mutant phenotypes will comprise analysis of microbial growth and hyphal development, quantification of adhesion to human oral epithelial cells and receptor-induced endocytosis.

The host response to wild type and mutated C. albicans will be investigated using a combination of cellular damage assays, analysis of intracellular signal transduction responses (EGFR phosphorylation, p38 MAPK signalling) and analysis of cytokine secretion from epithelial cells. The pathogenicity of the als3/ece1/sap2 triple null mutant will be evaluated using primary 3-D models of reconstituted human epithelium and established murine models of mucosal and systemic infection.

The findings of this research will contribute greatly to our understanding of the mechanisms that underpin C. albicans pathogenesis during mucosal and systemic infection.