GMP Synthase Is Required for Virulence Factor Production and Infection by Cryptococcus neoformans

Abstract

Edited by John M. DenuOver the last four decades the HIV pandemic and advances in medical treatments that also cause immunosuppression have produced an ever-growing cohort of individuals susceptible to opportunistic pathogens. Of these, AIDS patients are particularly vulnerable to infection by the encapsulated yeast Cryptococcus neoformans. Most commonly found in the environment in purine-rich bird guano, C. neoformans experiences a drastic change in nutrient availability during host infection, ultimately disseminating to colonize the purine-poor central nervous system. Investigating the consequences of this challenge, we have characterized C. neoformans GMP synthase, the second enzyme in the guanylate branch of de novo purine biosynthesis. We show that in the absence of GMP synthase, C. neoformans becomes a guanine auxotroph, the production of key virulence factors is compromised, and the ability to infect nematodes and mice is abolished. Activity assays performed using recombinant protein unveiled differences in substrate binding between the C. neoformans and human enzymes, with structural insights into these kinetic differences acquired via homology modeling. Collectively, these data highlight the potential of GMP synthase to be exploited in the development of new therapeutic agents for the treatment of disseminated, life-threatening fungal infections.In the past four decades there has been a dramatic escalation in the number of immunocompromised individuals (1). Although many of these are due to advances in immunosuppressive and chemotherapeutic technologies, the largest cohort is a direct result of the AIDS pandemic. Foreshadowed in 1981 with the occurrence of opportunistic Pneumocystis or human herpesvirus 8 infections in previously healthy individuals in Los Angeles (2, 3), AIDS and the lentivirus causing the disease soon gained broad public awareness (4 -6). With the advent of commercial blood testing, national blood bank screening programs were commenced in an effort to slow the spread of this emerging pandemic (7), and in 1987 the first treatment, zidovudine, marked what was thought to be an end to the crisis (8, 9). It was not, as was hoped, a miracle drug; the spread of AIDS continued across the globe and is now believed to have infected over 70 million people and killed 55 million to date (10).Since the discovery of the very first patients identified with Pneumocystis pneumonia, opportunistic fungal pathogens have been tightly linked with the AIDS pandemic. One of the key fungi often encountered in this context is Cryptococcus neoformans, a basidiomycete yeast responsible for cryptococcosis and a major cause of AIDS-related mortality (11). Most commonly found associated with purine-rich bird guano, spores or desiccated yeast cells from this environmental niche are inhaled into the lungs where, in an immunocompromised individual, the fungus can disseminate to the purine-poor central nervous system to cause meningoencephalitis.The fundamental treatment for cryptococcosis has not changed significantly ...

Helices on interdomain interface couple catalysis in the ATPPase domain with allostery inPlasmodium falciparumGMP synthetase

“…The lack of activity of the R539L mutant proves that the interdimer interactions are indispensable for PfGMPS catalysis. Unlike human GMPS and GMPS from higher eukaryotes which possess a 150 residue insertion in the dimerization domain that allows them to function as monomers, GMP synthetases from prokaryotes and certain eukaryotes including P. falciparum are obligate dimers [28,[38][39][40][41].…”

Cryptococcus neoformans ADS lyase is an enzyme essential for virulence whose crystal structure reveals features exploitable in antifungal drug design

“…Although antifungal agents that target purine biosynthesis have not yet been developed, there is increasing data that sug-gests this aspect of primary metabolism may be a druggable target for broad spectrum, non-toxic and affordable therapies to treat life-threatening fungal infections. Two GMP biosynthetic enzymes (IMP dehydrogenase and GMP synthase) and one ATP biosynthetic enzyme (ADS synthetase) have already been characterized at the genetic, enzymatic, and structural levels in C. neoformans as potential antifungal drug targets, and each have key active site differences compared with their human orthologs that could potentially enable the development of fungus-specific inhibitors (19,20,23). In contrast to these, the bifunctional nature of ADS lyase influences not only ATP biosynthesis, but also production of the key purine intermediate IMP (11); by disrupting IMP biosynthesis, the de novo production of both ATP and GTP is lost, suggesting that the inhibition of this enzyme may represent an even more powerful antifungal strategy.…”

“…Recent studies in the major fungal pathogens have highlighted the potential of purine biosynthetic enzymes as drug targets (19 -23). For example, the encapsulated yeast Cryptococcus neoformans has been shown to require IMP dehydrogenase, GMP synthase, ADS synthase, and AIR carboxylase to successfully infect a mammalian model (19,20,(23)(24)(25).…”

Classical and rational approaches to antifungal drug design

“…In contrast, the possible role of this enzyme as an antifungal target has not yet been investigated, nor has the structure in any member of the Kingdom fungi. Recent studies in the major fungal pathogens have highlighted the potential of purine biosynthetic enzymes as drug targets (14,15,82,83,258). For example, the encapsulated yeast Cryptococcus neoformans has been shown to require IMP dehydrogenase, GMP synthase, ADS synthase and AIR carboxylase to successfully infect a mammalian model (14,15,120,121,258).…”

“…Furthermore, the similarity makes the fungal enzyme an alternative model to understand the molecular basis of the effects of mutations leading to human ADS Lyase Deficiency disease. (14,15,258). In contrast to these, the bifunctional nature of ADS lyase influences not only ATP biosynthesis, but also production of the key purine intermediate IMP (250); by disrupting IMP biosynthesis, the de novo production of both ATP and GTP is lost, suggesting that the inhibition of this enzyme may represent an even more powerful antifungal strategy.…”