SEQUENCING THE MHC CLASS I PEPTIDES FROM TRYPANOSOMA CRUZI

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。克氏锥虫是一种胞内原生动物寄生虫，是查加斯病的病原体。据估计，拉丁美洲有1500万至2000万人感染这种破坏性疾病，这种慢性感染形式可表现为一种致命的心肌病。克氏毛滴虫无鞭毛体存在于宿主细胞的细胞质中，在那里它们释放的蛋白质可以进入主要组织相容性复合体(MHC)的处理和提呈途径。MHC-I类结合多肽和识别MHC-多肽复合体的CD8细胞毒性T淋巴细胞(CTL)是克氏毛滴虫细胞免疫的关键分子。因此，对感染宿主细胞表面MHC呈递的寄生虫肽的鉴定是疫苗研制的重要前奏。我们已经开发了一种替代策略来识别CTL反应的靶标，该策略使用病原体基因组的信息来避免产生CTL品系来进行肽鉴定的必要性。我们的方法不需要事先知道多肽的母体蛋白，也不要求基因组序列信息在本质上是完整的。在这里展示的例子中，基因组序列远远不完整。在实验上，该方法确实要求使用具有已知MHC结合基序的遗传特征模型，并且感兴趣的病原体也是已知的。这些数据表明，当CTL检测在实验上变得不可能时，使用二维分离与MS/MS和生物信息学相结合的严格自动测序方法是鉴定免疫学感兴趣的病原体基因产物的可行方法。这种方法可以提供一种系统的方法来识别各种传染病系统的亲本蛋白(疫苗候选)，而不依赖于生物检测。

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.