Molecular Mechanism of Entamoeba histolytica phagocytosis

描述（由申请人提供）：阿米巴病是第二大原生动物死亡原因。溶组织内阿米巴是一种肠道阿米巴，它通过吞噬结肠细菌和宿主细胞获得营养。溶组织性是侵袭性阿米巴病一个显著特征。尽管它的重要性，很少有人知道的机制，E。溶组织吞噬作用溶组织内阿米巴诱导宿主细胞凋亡，导致宿主细胞表面发生变化，引起未知的阿米巴吞噬受体。在哺乳动物中，胶原凝集素家族成员（例如，甘露糖结合凝集素（MBL））和结构相关的蛋白C1 q结合凋亡细胞，并通过其保守的胶原“尾”与钙网蛋白的相互作用启动吞噬作用。钙网蛋白，没有跨膜结构域，作为胶原凝集素和巨噬细胞受体CD 91之间的桥梁。初步数据表明：1）C1 q、MBL和collectin尾刺激E. 2）C1 q和MBL竞争结合E.钙网蛋白存在于阿米巴表面，并在与凋亡细胞相互作用期间重新定位于吞噬杯，4）人C1 q和凝集素MBL和SP-A竞争结合固定的阿米巴钙网蛋白，和5）特异性E.溶组织表面蛋白与钙网蛋白相互作用。因此，与凋亡细胞和细菌结合的宿主凝集素可以刺激E。通过其胶原尾部结构域与阿米巴受体的相互作用，钙网蛋白是一个合乎逻辑的候选者，如果钙网蛋白参与阿米巴的吞噬作用，它被假设这样做的聚集素和阿米巴表面上的钙网蛋白受体之间的桥接。具体目标验证了这些假设。在目标1中，将使用纯化的聚集素、C1 q和胶原聚集素“尾”来测试E. histolytica具有介导凋亡细胞吞噬的聚集蛋白尾受体。除了C1 q，collectins也调理粘膜分泌物中的细菌。因此，将进行研究以测试E.溶组织菌通过相同的机制吞噬凋亡细胞和细菌。目的2：利用RNA介导的干扰和另一种基因沉默方法来确定钙网蛋白是否参与E.溶组织吞噬作用重组钙网蛋白将用于结合研究，以确定是否E。histolytica具有钙网蛋白受体。在目标3中，将鉴定聚集蛋白或钙网蛋白受体。决定采取哪一项行动将取决于目标1和2的结果。在任何一种情况下，交联方法和互补的亲和力为基础的方法将结合质谱法，以确定相互作用的阿米巴表面蛋白。这些研究的成功完成将有助于对E. histolytica吞噬，并将大大增加我们的知识如何E。溶组织菌与宿主和结肠细菌相互作用。此外，这些研究对阿米巴病发病机制的新认识可能为其治疗和预防提出新的方法。 公共卫生相关性：溶组织内阿米巴是一种单细胞肠道寄生虫，可引起侵袭性阿米巴病（一种以血性腹泻和肝脓肿为特征的疾病），在侵入宿主组织时吞噬被杀死的细胞。本项目的目标是确定E.溶组织菌识别和吞噬死亡细胞的能力。这将为E.溶组织阿米巴引起侵袭性感染，可能提示治疗或预防阿米巴病的新方法。

DESCRIPTION (provided by applicant): Amebiasis ranks second as a protozoan cause of death. Entamoeba histolytica, the etiologic agent, is an intestinal ameba that acquires nutrients by phagocytosis of colonic bacteria, and phagocytosis of host cells by E. histolytica is a prominent feature of invasive amebiasis. Despite its importance, little is known about the mechanism of E. histolytica phagocytosis. Entamoeba histolytica induces host cell apoptosis, resulting in host cell surface changes that engage unknown amebic phagocytosis receptors. In mammals, collectin family members (e.g., mannose binding lectin (MBL)) and the structurally related protein C1q bind to apoptotic cells, and initiate phagocytosis by interaction of their conserved collagenous "tails" with calreticulin. Calreticulin, which has no transmembrane domain, serves as a bridge between collectins and the macrophage receptor CD91. Preliminary data show that: 1) C1q, MBL, and collectin tails stimulate E. histolytica phagocytosis, 2) C1q and MBL compete for binding to the surface of E. histolytica, 3) calreticulin is present on the amebic surface and re-localizes to the phagocytic cup during interaction with apoptotic cells, 4) human C1q and the collectins MBL and SP-A compete for binding to immobilized amebic calreticulin, and 5) specific E. histolytica surface proteins interact with calreticulin. Therefore, host collectins bound to apoptotic cells and bacteria may stimulate E. histolytica phagocytosis by interaction of their collagenous tail domain with an amebic receptor. Calreticulin is one logical candidate, and, if calreticulin participates in amebic phagocytosis, it is hypothesized to do so by bridging between collectins and a calreticulin receptor on the amebic surface. The specific aims test these hypotheses. In aim 1, purified collectins, C1q, and collagenous collectin "tails" will be used to test if E. histolytica has a receptor for the collectin tail that mediates engulfment of apoptotic cells. Except for C1q, the collectins also opsonize bacteria in mucosal secretions. Therefore, studies will be performed to test if E. histolytica engulfs apoptotic cells and bacteria via the same mechanism. In aim 2, RNA-mediated interference and an alternative gene silencing method will be used to determine if calreticulin participates in E. histolytica phagocytosis. Recombinant calreticulin will be used for binding studies to determine if E. histolytica has a calreticulin receptor. In aim 3, either a collectin or a calreticulin receptor will be identified. The decision of which to pursue will depend on the results from aims 1 and 2. In either case, cross-linking methods and complementary affinity-based methods will be combined with mass spectrometry to identify interacting amebic surface proteins. Successful completion of the proposed studies will provide a molecular understanding of E. histolytica phagocytosis, and will substantially augment our knowledge of how E. histolytica interacts with the host and with colonic bacteria. Furthermore, new insights into the pathogenesis of amebiasis that result from these studies may suggest novel methods for its treatment and prevention. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica, a single-celled intestinal parasite that causes invasive amebiasis (a disease Characterized by bloody diarrhea and liver abscesses), engulfs killed cells during invasion through host tissues. The goal of this project is to determine the molecular mechanisms underlying E. histolytica's ability to recognize and engulf killed cells. This will provide novel insights into how E. histolytica causes invasive infections, possibly suggesting new methods to treat or prevent amebiasis.