Targeted gene replacement demonstrates that myristoyl-CoA: protein N-myristoyltransferase is essential for viability of Cryptococcus neoformans.

Abstract

Cryptococcus neoformans is a major cause of systemic fungal infection in immunocompromised patients. Myristoyl-CoA:protein N-myristoyltransferase (Nmt) catalyzes the transfer of myristate (C14:0) from myristoyl-CoA to the N-terminal glycine of a subset of cellular proteins produced during vegetative growth of C. neoformans. A Glyw --Asp mutation was introduced into C. neoformans NMT by targeted gene replacement. The resulting strains are temperaturesensitive myristic acid auxotrophs. They are killed at 37C when placed in medium lacking myristate and, in an immunosuppressed animal model ofcryptococcal meningitis, are completely eliminated from the subarachnoid space within 12 days of initial infection. C. neoformans and human Nmts exhibit differences in their peptide substrate specificities. These differences can be exploited to develop a new class of fungicidal drugs.Cryptococcus neoformans var. neoformans is an opportunistic pathogen which has emerged as a serious cause of systemic fungal infection in immunocompromised humans (1-3). Persistent C. neoformans infections are common in AIDS patients after completion of primary therapy with amphotericin B or fluconazole for meningitis. This leads to relapse rates of 50-60%o and a shortened lifespan unless patients receive lifelong suppressive therapy, generally with fluconazole (4, 5). Both amphotericin B and fluconazole target ergosterol, the principal sterol in the organism's plasma membrane. Amphotericin B, a fungicidal macrolide, also binds to cholesterol in animal cell membranes, resulting in renal and other toxicities. Chronic suppressive therapy with the fungistatic drug fluconazole in severely immunocompromised hosts will most likely result in emergence of resistant strains (6). The search for alternative therapeutic targets in this organism would be helped by the ability to use targeted gene disruption to establish whether a given gene product is essential for viability. Protocols for high-efficiency transformation of C. neoformans have been reported (7-9). Gene replacement has remained elusive and may be strain-or locus-specific (9, 10).Myristoyl-CoA:protein N-myristoyltransferase (Nmt) catalyzes the cotranslational transfer of myristate (C14:0) from myristoyl-CoA to the amino-terminal glycine of a subset of eukaryotic cellular and viral proteins (11). Saccharomyces cerevisiae and human Nmts have an ordered bi-bi reaction mechanism: the apoenzymes bind to myristoyl-CoA, forming a high-affinity binary complex. Formation of this complex allows generation of a functional peptide binding site and subsequent generation of a myristoyl-CoA/Nmt/peptide ternary complex. After catalysis, CoA and then the myristoylpeptide product are released (12-14).NMTgenes have been isolated from S. cerevisiae, Candida albicans, Histoplasma capsulatum, C. neoformans, and humans (15-18). All are present in a single copy per haploid genome. Several S. cerevisiae N-myristoylproteins are essential for viability and depend upon a covalently bound myristoyl group for expression ...

Two N-Myristoyltransferase Isozymes Play Unique Roles in Protein Myristoylation, Proliferation, and Apoptosis

“…Because NMT activity is essential for the growth and survival of a variety of infectious organisms, NMT has been considered as a target for the development of new antibiotics (55,(58)(59)(60). For example, several studies have targeted the NMTs of Candida albicans and Cryptococcus neoformans, which are responsible for systemic fungal infections in immunocompromised humans, as treatment of such infections (43,(61)(62)(63)(64).…”

Protein myristoylation in health and disease

“…An immunosuppressed mouse model was used to show that C. albicans producing mutant NMT with greatly reduced activity did not kill the mice, unlike the wild-type NMT strain, suggesting a role in virulence. Similarly, it was shown that C. neoformans (which can cause chronic meningitis in immunosuppressed patients) possessing a less active mutant version of NMT cannot survive at 37°C without the addition of myristic acid to the medium [31]. The search for species-specific inhibitors has focused on the peptide binding pocket of NMT since this is not strictly conserved across species, unlike the myristoylCoA binding site (see Section NMT substrate specificity).…”

“…It appears to be ubiquitous in eukaryotes and has been isolated and characterised in yeast and fungi (Candida albicans, Saccharomyces cerevisiae [22], Cryptococcus neoformans [23] and Aspergillus nidulans [24]), parasitic protozoa (plasmodium species [25], Leishmania major [26] and, Trypanosoma brucei [26]), insects (Drosophila melanogaster [27]), plants (Arabidopsis thaliana [28]) and mammals (including mouse, rat, cow and human). NMT has been shown to be essential for the survival of S. cerevisiae [29], C. albicans [30], C. neoformans [31] and the bloodstream forms of the parasites L. major and T. brucei [32]. It is important in the development of Drosophila [27] and mice [33].…”

The Biology and Enzymology of ProteinN-Myristoylation

“…non-receptor tyrosine kinases (Dsrc42A, Dsrc64B). Finally, a NMT null allele causes lethality in the two principal causes of systemic fungal infections in immunocompromised humans, Candida albicans and Cryptococcus neoformans (26,27).…”