Genetic Interaction Analysis Reveals that Cryptococcus neoformans Utilizes Multiple Acetyl-CoA-Generating Pathways during Infection.

Cryptococcus neoformans is an important human fungal pathogen for which the external environment is its primary niche. Previous work has shown that two nonessential acetyl-CoA metabolism enzymes, ATP-citrate lyase () and acetyl-CoA synthetase (), play important roles in C. neoformans infection. Here, we took a genetic interaction approach to studying the interplay between these two enzymes along with an enzyme initially called but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-etoutyryl oA synthetase 1 () based on its biochemical activity and the systematic name of its substrate. and represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of with either or do not have significant synthetic phenotypes , although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the Δ Δ double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both and is increased relative to conditions. The Δ mutant is hypersusceptible to fluconazole despite its minimal phenotypes. These data not only provide insights into the mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment. The adaptation of environmental fungal pathogens to the mammalian host is critical to pathogenesis. Of these adaptations, the remodeling of carbon metabolism is particularly important. Here, we generated a focused set of double mutants of nonessential genes (, , ) involved in acetyl-CoA metabolism and examined their fitness and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of / was minimal . Consistent with these observations, the expression of and was increased relative to standard conditions. Furthermore, strains lacking both and were not viable. These data clearly show that C. neoformans employs multiple carbon metabolism pathways to adapt to the host environment. They also provide insights into the potential mechanism of action for anti-cryptococcal Acs inhibitors.