E. histolytica phagocytosis of apoptotic host cells

描述（由申请人提供）：拟议的培训计划结合了强化教学和基础实验室培训，为医学博士克里斯托弗·休斯顿在侵入性阿米巴病期间研究宿主-病原体生物学做好准备。溶组织内阿米巴原虫是阿米巴性结肠炎和肝脓肿的原生动物病因，估计每年有5000万人感染，造成多达10万人死亡。阿米巴病是一种惰性感染，由于组织破坏的程度，炎症是稀疏的。宿主细胞凋亡和吞噬是溶组织芽胞杆菌感染的进一步特征。事实上，临床医生使用红细胞吞噬术来区分溶组织芽胞杆菌和非致病性阿米巴异内阿米巴。这个项目的假设是，凋亡细胞的杀死和随后的快速吞噬限制了细胞内有毒物质的溢出，并以类似于多细胞生物中凋亡细胞的吞噬的方式防止炎症。因此，凋亡杀伤和吞噬作用可能使溶组织芽胞杆菌逃避宿主免疫反应而致病。休斯顿博士的初步研究证实了溶组织芽胞杆菌对宿主细胞的凋亡杀伤作用，并表明溶组织芽胞杆菌比健康细胞更有效地摄取凋亡细胞。特异性目的1是利用共聚焦显微镜和流式细胞术测定吞噬作用，确定触发阿米巴摄入的宿主细胞表面变化的性质和机制。特异性目的2是表征一种阿米巴ABC转运蛋白EhABC A1，它与秀丽隐杆线虫的吞噬相关转运蛋白ce -7同源。EhABC A1的功能/表达将被显性阴性突变体、反义RNA表达和反义肽核酸低聚物破坏。特异靶1的结果将阐明阿米巴细胞毒性的机制，并有助于阿米巴吞噬受体的鉴定。在Specific Aim 2中，成功干扰EhABC A1以测试其在吞噬作用中的作用将有助于确定阿米巴原虫的吞噬机制，并可能为缺乏吞噬功能的阿米巴原虫描述吞噬作用在体内毒力中的作用提供依据。

DESCRIPTION (provided by applicant): The proposed training plan combines intensive didactic and basic laboratory training to prepare Christopher Huston, M.D., for a career investigating host-pathogen biology during invasive amebiasis. Entamoeba histolytica, the protozoan cause of amebic colitis and liver abscess, infects an estimated 50 million people causing up to 100 thousand deaths annually. Amebiasis is an indolent infection and inflammation is sparse given the degree of tissue destruction. Apoptotic host cell killing and phagocytosis further characterize E. histolytica infection. Indeed, clinicians use erythrophagocytosis to distinguish E. histolytica from the non-pathogenic ameba Entamoeba dispar. This project's hypothesis is that apoptotic cell killing followed by rapid phagocytosis limits spillage of toxic intracellular contents and prevents inflammation in an analogous fashion to phagocytosis of apoptotic cells in multicellular organisms. In this way, apoptotic killing and phagocytosis are hypothesized to enable E. histolytica to evade the host immune response and cause disease. Preliminary studies by Dr. Huston have demonstrated apoptotic killing of host cells by E. histolytica and shown that E. histolytica ingested apoptotic cells more efficiently than healthy cells. Specific Aim 1 is to define the nature and mechanism of host cell surface changes that trigger amebic ingestion using confocal microscopy and flow cytometry to assay phagocytosis. Specific Aim 2 is to characterize an amebic ABC transporter, EhABC A1, with homology to the phagocytosis-associated transporter Ced-7 from Caenorhabditis elegans. EhABC A1 function/expression will be disrupted using dominant negative mutants, anti-sense RNA expression, and anti-sense peptide nucleic acid oligomers. Results from Specific Aim 1 will clarify the mechanism of amebic cytotoxicity as well as help to identify the amebic phagocytosis receptor. In Specific Aim 2, successful interference with EhABC A1 to test its role in phagocytosis will help to define the amebic phagocytosis machinery and may provide phagocytosis deficient amoebae to delineate the role of phagocytosis for in vivo virulence.