S3.4e Unraveling the role of DOG genes in a novel alternative pathway of glycerol biosynthesis in Candida albicans and its influence on virulence

Abstract

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMDOG genes, encoding for 2-deoxyglucose-6-phosphate phosphatase are low molecular weight phosphatases, with an unknown biological function. In contrast to Saccharomyces cerevisiae which has two DOG homologs, C. albicans only has one DOG gene. We hypothesized that DOG1 plays an important role under osmotic or ionic stress by biosynthesizing glycerol, which is known to be vital for biofilm formation and virulence of this pathogenic yeast, via a novel alternative pathway. The known classical pathway of glycerol production begins when the glycolytic intermediate molecule dihydroxyacetone phosphate (DHAP) is converted into glycerol-3-phosphate (G-3-P) by a pair of glycerol-3-phosphate-dehydrogenases, Gpd1 and Gpd2. G-3-P is further dephosphorylated into glycerol by glycerol-3-phosphate-phosphatases, Gpp1 and Gpp2. However, an alternative pathway, where DHAP is dephosphorylated into DHA, which is subsequently converted into glycerol has been proposed, but the enzymes involved in this process have not yet been described. We recently showed that in Saccharomyces cerevisiae, the DOG enzymes are involved in the production of DHA from DHAP, thereby allowing the synthesis of glycerol in the absence of the classical pathway. Overexpression of the DOG genes restored the osmo-tolerance of the gpp1Δ gpp2Δand gpd1Δ gpd2Δ double deletion strains. Furthermore, purified DOG enzymes could also convert DHAP into DHA (Awasthy et al., submitted). Since DOG1 has a potential role in biosynthesizing glycerol via an unconventional route, we are interested to determine its contribution in influencing virulence and biofilm formation in Candida albicans. This putative pathway has been overlooked for the past two decades, leaving behind an evident knowledge gap. We have now generated multiple deletion strains, using CRISPR Cas9, for the C. albicans counterparts of the GPP, GPD, and DOG genes as well as multiple DOG1 overexpression strains in which we observed the restoration of osmotic stress tolerance phenotypically and via growth curves. We also have NMR data showing the accumulation of various metabolites of central metabolism in these strains. Additionally, we have determined the possible role of DOG1 on virulence factors like morphogenesis, adhesion, and biofilm formation in vitro as well as in vivo, the latter with our catheter-based biofilm subcutaneous mouse model system. We also linked DOG1 and its role in glycerol biosynthesis to the survival of C. albicans inside macrophages. Finally, we would be setting up a high throughput small compound screening for this phosphatase as a potential antifungal drug target.