New tricks from old genes: Investigating G-protein signaling as a mediator of colony morphology and aerobic glycolysis in Aspergillus fumigatus

About This Grant

Aspergillus fumigatus is an environmental mould responsible for invasive pulmonary infections with an unacceptably high mortality rate. A critical determinant of the severity of invasive disease outcomes is the relative fitness of the infecting strain in low oxygen environments. Our laboratory has found that certain strains adopt a colony morphology, H-MORPH, associated with increased virulence and fitness in low oxygen. One mechanism that strains use to induce H-MORPH is by increasing expression of uncharacterized baf genes. H-MORPH strains also exhibit a unique metabolism that suggests the strains employ aerobic glycolysis. Mutants that overactivate G-protein signaling through gpaA also induce the H-MORPH colony morphology. As canonical regulators of glucose metabolism across fungal biology, G-proteins represents an attractive effector for baf genes to induce H-MORPH and aerobic glycolysis. The primary scientific objective of this proposal is to understand the molecular and metabolic mechanisms driving baf-mediated H-MORPH. I will do so by testing the following hypotheses: 1) that bafA relies on G-protein signaling to mediate H-MORPH, and 2) that H-MORPH strains adopt aerobic glycolysis. In Aim 1, I will employ CRISPR-Cas9 genetic techniques to explore the epistatic relationships between bafA, gpaA, G-protein coupled receptors, and H-MORPH. To complement these genetic approaches, I will employ proximity labeling of bafA to identify proteins in the molecular neighborhood of bafA, with a specific primary screening strategy prioritizing proteins involved in G-protein signaling. These approaches will provide me with training in complementary, mechanistic methods of exploring molecular hypotheses. In Aim 2, I will broaden my training in computational microbial metabolomics, to perform isotope labeling and subsequent GC/MS analysis of central fungal metabolites. This will allow calculation of the metabolic flux of H-MORPH strains relative to wildtype, testing the hypothesis that H-MORPH strains rewire their metabolism to de-prioritize oxidative phosphorylation in favor of glycolysis, fermentation, and the pentose phosphate pathway. To complement the metabolic flux analyses, I will define the glucose uptake and oxygen consumption of H-MORPH strains, with the expectation that, similar to Otto von Warburg’s observations in cancer cells over 100 years ago, H-MORPH strains consume less oxygen and increase their glucose consumption. Finally, I hypothesize that H- MORPH metabolism will have predictable influences on the growth of certain metabolic mutants. Our lab previously showed that a strain deficient in the cytochrome C oxidase (∆cycA) exhibits decreased growth. Consistent with our model of H-MORPH exhibiting decreased respiration, increasing bafA expression should have a noticeable increase in the growth of the ∆cycA strain. Overall, this proposal will provide me with the necessary training to characterize metabolic remodeling in A. fumigatus and the tools to mechanistically test molecular hypotheses as to how metabolic switches related to fungal virulence are regulated.