GENETIC-STUDIES REVEAL THAT MYRISTOYLCOA-PROTEIN N-MYRISTOYLTRANSFERASE IS AN ESSENTIAL ENZYME IN CANDIDA-ALBICANS

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 mu M myristate, Studies in an immunosuppressed-mouse model of C. albicans infection revealed that the NMT/Delta nmt strain produced 100% lethality within 7d 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.