Targeted gene replacement demonstrates that myristoyl-CoA: protein N-myristoyltransferase is essential for viability of Cryptococcus neoformans.
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 Gly487-->Asp mutation was introduced into C. neoformans NMT by targeted gene replacement. The resulting strains are temperature-sensitive myristic acid auxotrophs. They are killed at 37 degrees C when placed in medium lacking myristate and, in an immunosuppressed animal model of cryptococcal 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.
Lodge
J K
JK
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110.
Jackson-Machelski
E
E
Toffaletti
D L
DL
Perfect
J R
JR
Gordon
J I
JI
D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000254:growth & development / Q000472:pathogenicity; D017931:DNA Primers; D005821:Genetic Techniques; D005998:Glycine; D006801:Humans; D007700:Kinetics; D016919:Meningitis, Cryptococcal / Q000382:microbiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013346:Subarachnoid Space / Q000382:microbiology; D013379:Substrate Specificity; D013696:Temperature
D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase; D005998:Glycine