SEQUENCING THE MHC CLASS I PEPTIDES FROM TRYPANOSOMA CRUZI

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Trypanosoma cruzi is an intracellular protozoan parasite and the causative agent of Chagas' disease. This devastating disease affects an estimated 15 to 20 million persons in Latin America, and the chronic form of the infection can manifest itself as a fatal cardiomyopathy. T. cruzi amastigotes reside in the cytoplasm of host cells where they release proteins that can enter the major histocompatibility complex (MHC) processing and presentation pathway. MHC class I-bound peptides and the CD8 cytotoxic T lymphocytes (CTLs) that recognize the MHC-peptide complex are critical players in cell-mediated immunity to T. cruzi. For this reason, the identification of parasite peptides that are presented by the MHC on the surface of infected host cells is an important prelude to the development of vaccines. We have developed an alternative strategy for identifying targets of the CTL response that use information from the pathogen's genome to avoid the necessity of generating CTL lines for peptide identification. Our approach does not necessitate that the parent protein of the peptide be known in advance, or that genome sequence information be complete in nature. In the example presented here, the genome sequence was far from complete. Experimentally, the method does demand that genetically characterized models with a known MHC binding motif be used and that the pathogen of interest is also known. These data suggest that a rigorous automated sequencing approach employing two-dimensional separations in conjunction with MS/MS and bioinformatics is a feasible approach to identify pathogen gene products of immunological interest when CTL assay is rendered experimentally impossible. Such an approach may provide a systematic method to identify parent proteins (vaccine candidates), independent of biological assays, for a variety of infectious disease systems.