Molecular Mechanism of Entamoeba histolytica phagocytosis

描述（由申请人提供）：阿米巴病在原生动物死因中排名第二。病原溶组织内阿米巴是一种通过吞噬结肠细菌来获取营养的肠道阿米巴，而溶组织内阿米巴吞噬宿主细胞是侵袭性阿米巴病的一个显著特征。尽管它很重要，但对溶组织芽胞杆菌吞噬作用的机制知之甚少。溶组织内阿米巴诱导宿主细胞凋亡，导致宿主细胞表面变化，参与未知阿米巴吞噬受体。在哺乳动物中，集合家族成员（如甘露糖结合凝集素（MBL））和结构相关蛋白C1q结合到凋亡细胞上，并通过其保守的胶原“尾巴”与钙网蛋白的相互作用启动吞噬。钙网蛋白不具有跨膜结构域，是巨噬细胞受体CD91与集合蛋白之间的桥梁。初步研究结果表明：1)C1q、MBL和集合蛋白可刺激溶组织芽胞杆菌的吞噬；2)C1q和MBL可竞争结合到溶组织芽胞杆菌的表面；3)钙网蛋白存在于阿米巴表面，并在与凋亡细胞相互作用时重新定位到吞噬杯中；4)人C1q与集合蛋白MBL和SP-A竞争结合到固定的阿米巴钙网蛋白上；5)特异性溶组织芽胞杆菌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集合素可能通过其胶原尾结构域与阿米巴受体的相互作用刺激溶组织芽胞杆菌吞噬。钙网蛋白是一个合乎逻辑的候选者，而且，如果钙网蛋白参与阿米巴的吞噬作用，假设它是通过在阿米巴表面的集合素和钙网蛋白受体之间架桥来完成的。具体目标验证了这些假设。在目的1中，纯化的集合素、C1q和胶原集合“尾部”将被用于测试溶组织芽孢杆菌是否具有介导凋亡细胞吞噬的集合尾部受体。除C1q外，这些集合物还能调理粘膜分泌物中的细菌。因此，我们将进一步研究溶组织芽胞杆菌是否通过同样的机制吞噬凋亡细胞和细菌。在目的2中，rna介导的干扰和另一种基因沉默方法将用于确定钙调蛋白是否参与溶组织芽胞杆菌的吞噬。重组钙网蛋白将用于结合研究，以确定溶组织芽胞杆菌是否具有钙网蛋白受体。在目标3中，要么是一个集合或钙调蛋白受体将被识别。选择哪一个将取决于目标1和目标2的结果。在任何一种情况下，交联方法和基于互补亲和力的方法将与质谱法相结合，以鉴定相互作用的阿米巴表面蛋白。这项研究的成功完成将提供对溶组织芽胞杆菌吞噬作用的分子理解，并将大大增加我们对溶组织芽胞杆菌如何与宿主和结肠细菌相互作用的认识。此外，这些研究对阿米巴病发病机制的新见解可能为其治疗和预防提供新的方法。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（一种以带血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。该项目的目标是确定溶组织芽胞杆菌识别和吞噬死亡细胞的能力的分子机制。这将为了解溶组织芽胞杆菌如何引起侵袭性感染提供新的见解，可能提出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.