This Research Career Award application describes a mentored research program for the candidate, Dr. Mylonakis, in fungal pathogenesis. The candidate is trained in Infectious Diseases and proposes a research program to study virulence of Cryptococcus neoformans. The candidate's sponsor, Dr. Calderwood, has a Iongtime interest in using simple organisms as model hosts for the study of human pathogens, while the candidate's cosponsor, Dr. Heitman, has a Iongtime interest in the genetics of C. neoformans and has developed a number of related molecular biology protocols. The candidate has developed a novel system for studying genetic and molecular mechanisms of C. neoformans pathogenesis using the nematode model organism Caenorhabditis elegans. He has found that C. neoformans kills C. elegans, and several genes, such as those associated with signal transduction pathways, laccase production and the alpha mating type, previously shown to be involved in mammalian virulence, also play a role in C. elegans killing. He used this system to screen a bank of randominsertional mutants that were developed in the C. neoformans H99 background. From 350 mutants tested, 7 were identified as attenuated in C. elegans, with the hypovirulence persisting after the mutation was crossed to a clean genetic background. Genetic analysis of the first mutant revealed that the mutation occurred in a gene homologous to KIN1 of Saccharomyces cerevisiae.
The SPECIFIC AIMS are as follows: 1) to develop and use the C. elegans system to screen a library of random C. neoformans insertional mutants and then to identify the disrupted genes and utilize a pathogenic wild type strain to make defined deletion mutations in each of the genes identified and develop the corresponding reconstituted strains, 2) to use the tail vein and lung inhalation murine models to test if the genes identified through the screen in C. elegans are involved in mammalian virulence, 3) to perform in-depth analysis for a limited number of selected mutants that demonstrate the most dramatic hypovirulence in both C. elegans and mice. The analysis will include evaluation of capsule, melanin production and mating. The research plan proposed with focus on the use of C. elegans as a facile model to study basic, evolutionarily conserved pathways associated with cryptococcal infection. Because C. neoformanshas similarities with other pathogenic yeasts, we expect that our findings will facilitate the study of fungus/host interaction in general.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08AI063084-01
Application #
6859552
Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
Program Officer
Duncan, Rory A
Project Start
2005-07-01
Project End
2008-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
1
Fiscal Year
2005
Total Cost
$125,280
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Means, Terry K; Mylonakis, Eleftherios; Tampakakis, Emmanouil et al. (2009) Evolutionarily conserved recognition and innate immunity to fungal pathogens by the scavenger receptors SCARF1 and CD36. J Exp Med 206:637-53
Peleg, Anton Y; Monga, Divya; Pillai, Satish et al. (2009) Reduced susceptibility to vancomycin influences pathogenicity in Staphylococcus aureus infection. J Infect Dis 199:532-6
Peleg, Anton Y; Jara, Sebastian; Monga, Divya et al. (2009) Galleria mellonella as a model system to study Acinetobacter baumannii pathogenesis and therapeutics. Antimicrob Agents Chemother 53:2605-9
Pukkila-Worley, Read; Mylonakis, Eleftherios (2008) Epidemiology and management of cryptococcal meningitis: developments and challenges. Expert Opin Pharmacother 9:551-60
Peleg, Anton Y; Tampakakis, Emmanouil; Fuchs, Beth Burgwyn et al. (2008) Prokaryote-eukaryote interactions identified by using Caenorhabditis elegans. Proc Natl Acad Sci U S A 105:14585-90
Mylonakis, Eleftherios; Casadevall, Arturo; Ausubel, Frederick M (2007) Exploiting amoeboid and non-vertebrate animal model systems to study the virulence of human pathogenic fungi. PLoS Pathog 3:e101
Fuchs, Beth Burgwyn; Tang, Robin J; Mylonakis, Eleftherios (2007) The temperature-sensitive role of Cryptococcus neoformans ROM2 in cell morphogenesis. PLoS One 2:e368
Reese, Amy J; Yoneda, Aki; Breger, Julia A et al. (2007) Loss of cell wall alpha(1-3) glucan affects Cryptococcus neoformans from ultrastructure to virulence. Mol Microbiol 63:1385-98
Aperis, George; Fuchs, Beth Burgwyn; Anderson, Christine A et al. (2007) Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain. Microbes Infect 9:729-34
Fuchs, Beth Burgwyn; Tegos, George P; Hamblin, Michael R et al. (2007) Susceptibility of Cryptococcus neoformans to photodynamic inactivation is associated with cell wall integrity. Antimicrob Agents Chemother 51:2929-36

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