Candida glabrata ace2 hypervirulence: Looking at the genes behind the effect

Abstract

Our current understanding of virulence in the opportunistic pathogen Candida glabrata is in its infancy. To expand our understanding of C. glabrata virulence, an investigation into the genes transcriptionally regulated by the first virulence-moderating gene to be discovered in Candida, ACE2, was undertaken. For the first time in C. glabrata, a partial Ace2p regulon containing 12 previously univestigated genes that show Ace2p­‐dependent expression has been defined. Study of their promoter regions discovered possible Ace2p DNA binding sites, with 10 genes showing possible direct regulation by Ace2p in C. glabrata. A reductionist approach was employed in an effort to study the effects of the deletion of each of the 12 partial Ace2p-­regulon genes in C. glabrata individually. Both phenotypic and virulence analyses were used to analyse the results of the individual deletions with regard to the extreme hypervirulence displayed by the ace2 null cells. The results indicate that out of the 10 genes successfully disrupted, six were shown to be significantly different when compared with the C. glabrata wild type in a neutropenic murine model of systemic candidiasis and therefore hypervirulent. Each of the six hypervirulent genes showed 100% mortality after five days post infection, markedly different to the hypervirulence displayed by the ace2 null, which displayed 100% mortality within 18 hours post infection, and to the wild type, which displayed 40% mortality after 14 days post infection. The C. glabrata sic1 null was also found to elicit significantly increased cytokine release in an in vitro macrophage infection assay, as measured by IL­‐6 and TNFα ELISA. The results presented in this thesis therefore indicate that the hypervirulence and immune over-stimulation shown by the ace2 mutant in C. glabrata is most likely multifactorial with a combination of antivirulence genes contributing to the extreme hypervirulence and immune over-stimulation. During the course of investigation into the Ace2p regulon, a novel gene lacking homology to putative glucanases or other yeast genes was highlighted. Therefore the uncharacterised Ace2p regulon gene DSE1, whose function is currently unknown, was investigated. Using C. glabrata and its close genetic relative, the model organism Saccharomyces cerevisiae, a study of the localisation, phenotype and structural prediction revealed a possible enzymatic or regulatory function for Dse1p, localised to the cell budding area and daughter cell, with a likely role in cell wall metabolism and cell separation within C. glabrata.