Cruz MC, Cavallo L, Heitman J; American Society for Microbiology. General Meeting.
Abstr Gen Meet Am Soc Microbiol. 1999 May 30-Jun 3; 99: 319 (abstract no. F-119).
Duke Univ. Medical Ctr, Durham, NC.
Background: Cryptococcus neoformans is a fungal pathogen that causes life-threatening central nervous system infections in patients immunocompromised by AIDS, chemotherapy, organ transplantion, or high dose steroids. Current antifungal drug therapies are limited and suffer from toxic side effects and drug resistance. Previous studies in our lab established that C. neoformans is sensitive in vitro to the immunosuppressive agent rapamycin. In the yeast Saccharomyces cerevisiae and in T-cells, rapamycin forms complexes with the FKBP12 prolyl isomerase that block cell-cycle progression by inhibiting the TOR kinases. Hypothesis. In this study we tested the hypothesis that a TOR homolog is the target of rapamycin in C. neoformans. Experimental Methods and Results: Here, we report the cloning of a C. neoformans TOR1 homolog by degenerate PCR and a subsequent screen of a C. neoformans genomic library. The open reading frame of the C. neoformans TOR1 gene encodes a 2360 amino acid protein. The C-terminal region of the TOR1 protein contains the conserved FRB (FKBP12-rapamycin binding) and kinase domains. The C. neoformans TOR1 kinase domain shares 78%, 77%, and 73% identity to the S. cerevisiae TOR1, TOR2, and mammalian mTOR proteins respectively. We have identified a spontaneous mutation in the TOR1 FRB domain (serl846leu) that confers rapamycin resistance. Furthermore, in a two-hybrid assay, rapamycin dependent interactions were detected between wild- type C. neoformans TOR1 and cryptococcal FKBP12, but not between serl846leu mutant TOR1 and the FKBP12-rapamycin complex. Conclusion: We have identified a C. neoformans TOR1 homolog and characterized a TOR1 mutation that confers rapamycin resistance in C. neoformans by preventing FKBP12- rapamycin binding to TOR1 confirming that TOR1 is the target of rapamycin in C. neoformans.
Publication Types:
Keywords:
- Antigens, Fungal
- Cryptococcus neoformans
- Humans
- In Vitro
- Protein Kinases
- Saccharomyces cerevisiae
- Saccharomyces cerevisiae Proteins
- Sirolimus
- Tacrolimus Binding Protein 1A
- Yeasts
- immunology
- mTOR protein
Other ID:
UI: 102195712
From Meeting Abstracts