Targeted gene replacement demonstrates that myristoyl-CoA: protein N-myristoyltransferase is essential for viability of Cryptococcus neoformans
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J K Lodge1, E Jackson-Machelski, D L Toffaletti
1Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110.
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 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.
Insights
Targeting Cryptococcus neoformans Nmt with a specific mutation creates a temperature-sensitive strain dependent on myristic acid. This discovery offers a novel strategy for developing new antifungal drugs against serious fungal infections.
Area of Science:
- Mycology
- Molecular Biology
- Infectious Diseases
Background:
- Cryptococcus neoformans causes systemic fungal infections, particularly in immunocompromised individuals.
- Myristoyl-CoA:protein N-myristoyltransferase (Nmt) is crucial for modifying cellular proteins in C. neoformans.
- Nmt catalyzes the transfer of myristate to specific proteins during fungal growth.
Purpose of the Study:
- To investigate the role of C. neoformans Nmt in fungal viability.
- To develop a genetically modified strain of C. neoformans with conditional lethality.
- To explore Nmt as a potential target for novel antifungal therapies.
Main Methods:
- Introduction of a Gly487-->Asp mutation into the C. neoformans NMT gene via targeted gene replacement.
- Characterization of the resulting mutant strains as temperature-sensitive myristic acid auxotrophs.
- Evaluation of mutant strain efficacy in an immunosuppressed animal model of cryptococcal meningitis.
Main Results:
- The Gly487-->Asp mutation rendered C. neoformans temperature-sensitive and dependent on myristic acid for survival.
- Mutant strains were killed at 37°C in the absence of myristate.
- Complete elimination of the mutant pathogen was observed in the subarachnoid space of infected animals within 12 days.
Conclusions:
- The study successfully created a conditional lethal mutant of C. neoformans by targeting Nmt.
- Differences in substrate specificity between C. neoformans and human Nmt present an opportunity for selective drug development.
- Targeting Nmt offers a promising avenue for developing a new class of fungicidal drugs against C. neoformans infections.