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 (ACL1) and acetyl-CoA synthetase (ACS1), 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 ACS2 but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-ketobutyryl CoA synthetase 1 (KBC1) based on its biochemical activity and the systematic name of its substrate. ACL1 and ACS1 represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of KBC1 with either ACS1 or ACL1 do not have significant synthetic phenotypes in vitro, although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the acs1Δ kbc1Δ double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log10 CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both ACS1 and KBC1 is increased in vivo relative to in vitro conditions. The acs1Δ mutant is hypersusceptible to fluconazole in vivo despite its minimal in vitro phenotypes. These data not only provide insights into the in vivo mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment. IMPORTANCE 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 (ACL1, ACS1, KBC1) involved in acetyl-CoA metabolism and examined their fitness in vitro and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of ACS1/KBC1 was minimal in vitro. Consistent with these observations, the expression of ACS1 and KBC1 was increased in vivo relative to standard in vitro conditions. Furthermore, strains lacking both ACL1 and ACS1 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.

Gluconate Kinase Is Required for Gluconate Assimilation and Sporulation in Cryptococcus neoformans.

Cryptococcus neoformans is an environmental yeast and an opportunistic human pathogen. The ability to cause disease depends on the ability to adapt to the human host. Previous studies implicated infectivity-related kinase 3 (IRK3, CNAG_03048) as required for establishing an infection. We genetically and biochemically characterized IRK3 as a gluconate kinase and propose the name GNK1. This metabolic enzyme utilizes gluconate to produce 6-phosphogluconate as part of the alternative oxidative phase of the pentose phosphate pathway (AOXPPP). The presence of GNK1 confirms that the AOXPPP is present and able to compensate for loss of the traditional OXPPP, providing an explanation for its nonessentiality. C. neoformans can utilize gluconate as an alternative carbon source in a GNK1-dependent manner. In our efforts to understand the role of GNK1 in host adaptation and virulence, we found that GNK1-deficient mutants have variable virulence and carbon dioxide tolerance across multiple strains, suggesting that second site mutations frequently interact with GNK1 deletion mutations. In our effort to isolate these genetic loci by backcrossing experiments, we discovered that GNK1-deficient strains are unable to sporulate. These data suggest that gluconate metabolism is critical for sporulation of C. neoformans. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that contributes to nearly 180,000 deaths annually. We characterized a gene named GNK1 that appears to interact with other genetic loci involved with the ability of C. neoformans to act as a pathogen. While these interacting genetic loci remain elusive, we discovered that GNK1 plays roles in both metabolism and mating/sporulation. Further interrogation of the mechanistic role for GNK1 in sexual reproduction may uncover a larger network of genes that are important for host adaptation and virulence.

Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.