SEQUENCING THE MHC CLASS I PEPTIDES FROM TRYPANOSOMA CRUZI

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。克氏锥虫是一种细胞内的原生动物寄生虫，是南美锥虫病的病原体。这一毁灭性疾病影响到拉丁美洲估计1 500万至2 000万人，慢性感染可表现为致命的心肌病。T.克氏无鞭毛体存在于宿主细胞的细胞质中，在那里它们释放可以进入主要组织相容性复合体（MHC）加工和呈递途径的蛋白质。MHC I类结合肽和识别MHC-肽复合物的CD 8细胞毒性T淋巴细胞（CTL）是细胞介导的T细胞免疫的关键参与者。克鲁兹由于这个原因，鉴定由感染的宿主细胞表面上的MHC呈递的寄生虫肽是疫苗开发的重要前奏。我们已经开发了一种用于鉴定CTL应答的靶的替代策略，其使用来自病原体基因组的信息以避免产生用于肽鉴定的CTL系的必要性。我们的方法不需要预先知道肽的亲本蛋白，或者基因组序列信息在自然界中是完整的。在本例中，基因组序列远未完成。实验上，该方法确实要求使用具有已知MHC结合基序的遗传表征模型，并且感兴趣的病原体也是已知的。这些数据表明，一个严格的自动化测序方法，采用二维分离结合MS/MS和生物信息学是一种可行的方法来确定免疫学感兴趣的病原体基因产物时，CTL试验是实验上不可能的。这种方法可以为各种传染病系统提供一种独立于生物测定的系统性方法来鉴定亲本蛋白（候选疫苗）。

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.