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密集的教学和基础实验室培训，为医学博士Christopher Huston在侵袭性阿米巴病期间研究宿主病原体生物学做好准备。溶组织内阿米巴是引起阿米巴结肠炎和肝脓肿的原生动物，估计每年感染5000万人，造成多达10万人死亡。阿米巴病是一种迟发性感染，考虑到组织破坏的程度，炎症是稀少的。宿主细胞的凋亡杀伤和吞噬作用进一步说明了溶组性肠杆菌感染的特征。事实上，临床医生使用红细胞吞噬作用来区分组织溶解埃希菌和非致病的阿米巴。该项目的假设是，在快速吞噬之后的凋亡细胞杀死限制了有毒细胞内内容物的溢出，并以类似于多细胞生物体中吞噬凋亡细胞的方式防止炎症。通过这种方式，假设了细胞凋亡和吞噬作用，使溶组织埃希菌能够逃避宿主的免疫反应，从而导致疾病。休斯顿博士的初步研究已经证明了溶组织肠杆菌对宿主细胞的凋亡杀伤作用，并表明溶组织肠杆菌比健康细胞更有效地摄取凋亡细胞。具体目标1是利用共聚焦显微镜和流式细胞术检测吞噬功能，以确定引起阿米巴摄取的宿主细胞表面变化的性质和机制。特异目的2是鉴定与秀丽线虫吞噬相关转运蛋白Ced-7同源的阿米巴ABC转运蛋白EhABC A1。EhABC A1的功能/表达将被显性负性突变体、反义RNA表达和反义肽核酸寡聚体破坏。特异性靶点1的结果将有助于阐明阿米巴细胞毒性的机制，并有助于鉴定阿米巴吞噬受体。在特定目的2中，成功干扰EhABC A1以测试其在吞噬中的作用将有助于确定阿米巴吞噬机制，并可能提供吞噬缺陷的阿米巴来描述吞噬作用在体内毒力中的作用。

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.