Molecular Mechanism of Entamoeba histolytica phagocytosis

描述(申请人提供)：阿米巴病在原生动物死亡原因中排名第二。溶组织内阿米巴是一种肠道阿米巴，通过吞噬结肠细菌获得营养，其吞噬宿主细胞是侵袭性阿米巴病的显著特征。尽管它很重要，但人们对溶组织性肠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，导致宿主细胞表面发生改变，与未知的阿米巴吞噬受体结合。在哺乳动物中，凝集素家族成员(如甘露糖结合凝集素(MBL))和结构相关的蛋白C1q与凋亡细胞结合，并通过其保守的胶原蛋白尾巴与钙网蛋白相互作用启动吞噬功能。钙网蛋白没有跨膜区，是集合素和巨噬细胞受体CD91之间的桥梁。初步数据表明：1)C1q、MBL和集合素尾巴刺激溶组织埃希氏菌吞噬功能，2)C1q和MBL竞争结合到溶组织埃希氏菌表面，3)钙网织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与集合素MBL和SP-A竞争结合固定的阿米巴钙网蛋白，5)特异的溶组织埃希氏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性尾部结构域与阿米巴受体相互作用来刺激溶组织埃希菌的吞噬作用。钙网织蛋白是一个合理的候选者，如果钙网织蛋白参与阿米巴吞噬作用，它被认为是通过集合素和阿米巴表面的钙网织蛋白受体之间的桥梁来实现的。具体的目标验证了这些假设。在目标1中，纯化的集合素、C1q和胶原性集合素“尾巴”将被用来测试溶组埃希氏菌是否有介导吞噬凋亡细胞的集合素尾巴的受体。除了C1q，这些集合素还能调理粘膜分泌物中的细菌。因此，将进行研究，以检验溶组织埃希氏菌是否通过相同的机制吞噬凋亡的细胞和细菌。在目标2中，将使用RNA介导的干扰和一种替代的基因沉默方法来确定钙网织蛋白是否参与溶组织埃希氏菌的吞噬作用。重组钙网织蛋白将用于结合研究，以确定溶组肠杆菌是否具有钙网织蛋白受体。在目标3中，将鉴定集合素或钙网状蛋白受体。选择哪种方法将取决于目标1和目标2的结果。在这两种情况下，交联法和基于互补亲和力的方法都将与质谱学相结合，以鉴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从分子水平上了解溶组织性肠杆菌的吞噬作用，并将大大增强我们对溶组织性肠杆菌与宿主和结肠菌相互作用的认识。此外，这些研究对阿米巴病发病机制的新见解可能会为其治疗和预防提供新的方法。与公共卫生相关：溶组织内阿米巴是一种单细胞肠道寄生虫，导致侵袭性阿米巴病(一种以血性腹泻和肝脓肿为特征的疾病)，在通过宿主组织入侵期间吞噬被杀死的细胞。该项目的目标是确定溶组织埃希菌识别和吞噬死亡细胞能力的分子机制。这将为了解溶组织埃希氏菌如何引起侵袭性感染提供新的见解，可能会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.