Genetic studies reveal that myristoylCoA:protein N‐myristoyltransferase is an essential enzyme in Candida albicans

Abstract

MyristoylCoA:protein N-myristoyltransferase (Nmt) catalyses the co-translational, covalent attachment of myristate (C14:0) to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction. The NMT1 gene is essential for vegetative growth of Saccharomyces cerevisiae. Studies were carried out to determine if Nmt is also essential for vegetative growth of the pathogenic fungus Candida albicans. A strain of C. albicans was constructed in which one copy of NMT was partially deleted and disrupted. A Gly-447-->Asp mutation was introduced into the second NMT allele. This mutation produced marked reductions in catalytic efficiency at 24 and 37 degrees C, as judged by in vitro kinetic studies of the wild-type and mutant enzymes which had been expressed in, and purified from, Escherichia coli. The growth characteristics of isogenic NMT/NMT, NMT/delta nmt, and nmt delta/nmtG447D C. albicans strains were assessed under a variety of conditions. Only the nmt delta/nmtG447D strain required myristate for growth. This was true at both 24 and 37 degrees C. Palmitate could not substitute for myristate. Incubation of nmt delta/nmtG447D cells at 37 degrees C in the absence of myristate resulted in cell death as observed by the inability to form colonies on media supplemented with 500 microM myristate. Studies in an immunosuppressed-mouse model of C. albicans infection revealed that the NMT/delta nmt strain produced 100% lethality within 7 d after intravenous administration while the isogenic nmt delta/nmtG447G strain produced no deaths even after 21 d. These observations establish that Nmt is essential for vegetative growth of C. albicans and suggest that inhibitors of this acyltransferase may be therapeutically useful fungicidal agents.

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).…”

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).…”

N-Myristoylation of Arf proteins in Candida albicans: an in vivo assay for evaluating antifungal inhibitors of myristoyl-CoA:protein N-myristoyltransferase

“…Comparable values were noted 4 h after withdrawal of myristate (data not shown). It is important to note that nmt447DlnmtA cells require supplementation of YPD with myristate for growth at 24 "C and begin to die within 4 h after removal of myristate (Weinberg et al, 1995).…”

“…Withdrawal of myristate from the medium results in cell death, thereby establishing that Nmt is essential in these two organisms. In addition, an immunosuppressed rabbit model of human C. neoformans meningitis and an immunosuppressed mouse model of systemic C. albicans infection were used, together with isogenic strains of fungi with either wildtype or the conditional lethal nmt allele, to demonstrate that (genetic) attenuation of Nmt activity allows the immunocompromised host to cure itself of the fungal infection (Lodge et at., 1994b;Weinberg et al, 1995).…”

“…Nmt is encoded by a single copy gene in Candida albicans and Cryptococcus neoformans (Wiegand et al, 1992;Lodge et al, 1994a). Conditional lethal nmt alleles have been introduced into C. albicans and C. neoformans genomes by homologous recombination (Weinberg et al, 1995;Lodge et al, 1994b). These alleles specify Nmts with reduced affinity for myristoyl-CoA owing to substitution of a conserved Gly, located five amino acids from their C-terminus, with an Asp.…”