Function and Vaccine Potential of a Clostridium difficile Type IV Pilus Subunit

描述（由申请方提供）：艰难梭菌是一种革兰氏阳性、孢子形成、专性厌氧菌，已成为美国医院内腹泻的主要原因。细菌感染有多种表现，从无症状的结肠定植到大量腹泻、伪膜性结肠炎和死亡。感染的抗生素治疗是可用的，尽管治疗可能失败，患者可能复发。其他可用的治疗方法包括粪便移植，在非常严重的情况下，结肠切除术。C.艰难梭菌感染每年导致14，000人死亡;因此，迫切需要更好地了解其发病机制以及改进预防措施。虽然C.艰难梭菌仍在研究中，来自其他肠道病原体的证据表明，附着于上皮细胞是发病机制中的必要步骤。IV型菌毛（T4 P）是一种用于粘附和运动的细菌附属物，最近在C. difficile基因组T4 P的主体由菌毛蛋白组成;主要结构亚基是主要菌毛蛋白，而其他类似结构的蛋白质称为次要菌毛蛋白或菌毛蛋白样蛋白，以较低的丰度存在，并在菌毛生物发生中具有各种作用。梭艰难梭菌基因组编码9种特异性菌毛蛋白或菌毛蛋白样蛋白。该建议侧重于功能和疫苗潜力 一种叫PilJ的蛋白质我假设PilJ是一种次要的菌毛蛋白，并且其丰度低于预测的主要菌毛蛋白PilA 1;我将使用定量免疫印迹和qRT-PCR来验证这一假设。此外，我假设PilJ是T4 P粘附素，并且它与宿主细胞上的特异性受体相互作用。为了鉴定这种受体，我将采用两种不同的方法;一种是将菌毛蛋白与其受体交联，另一种是利用人类单倍体细胞系。最后，我假设用PilJ和佐剂免疫将对C.艰难梭菌的小鼠模型中。艰难感染 用PilJ免疫小鼠和兔子产生对蛋白质的强烈和特异性反应;我预测这种反应将是保护性的。本实验的结局为死亡率、发病率、组织病理学和终末抗体滴度。这些结果将阐明PilJ在C.艰难的T4 P和T4 P与宿主之间的相互作用。此外，它们为C提供了一个令人兴奋的新目标。艰难梭菌疫苗

DESCRIPTION (provided by applicant): Clostridium difficile is a Gram-positive, spore-forming, obligate anaerobe that has become the leading cause of nosocomial diarrhea in the United States. Infection with the bacterium has a variety of manifestations, ranging from asymptomatic colonization of the colon to copious diarrhea, pseudomembranous colitis, and death. Antibiotic treatment for infection is available, although treatment can fail and patients can relapse. Other available therapies include fecal transplant and, in very serious cases, colectomy. C. difficile infection results in 14,000 deaths per year; thus, a better understanding of its pathogenesis as well as improved preventative measures are urgently necessary. Although the exact sequence of events in initial colonization with C. difficile is still under investigation, evidence from othr intestinal pathogens suggests that attachment to epithelial cells is a requisite step in pathogenesis. Genes for components of a Type IV pilus (T4P), a type of bacterial appendage used in adhesion and motility, were recently identified in the C. difficile genome. The main body of the T4P is composed of pilins; the major structural subunit is the major pilin, whereas other, similarly structured proteins termed minor pilins or pilin-like proteins, are present at lower abundances and have various roles in pilus biogenesis. The C. difficile genome encodes nine specific pilins or pilin-like proteins. This proposal focuses on the function and vaccine potential of one such protein, PilJ. I hypothesize that PilJ is a minor pilin, and is present at a lower abundance than a predicted major pilin, PilA1; I will use quantitative immunoblotting and qRT-PCR to test this hypothesis. Furthermore, I hypothesize that PilJ is the T4P adhesin, and that it interacts with a specific receptor on the host cell. To identify this receptor, I will take two different approaches; one involves cross-linking the pilin to its receptor, while the other makes use of a human haploid cell line. Finally, I hypothesize that immunization with PilJ and adjuvant will be protective against challenge with C. difficile in a murine model of C. difficile infection. Immunization of mice and rabbits with PilJ produces a strong and specific response to the protein; I predict that this response will be protective. Outcomes for this experiment will be mortality, morbidity, histopathology, and terminal antibody titer. These results will illuminate th role of PilJ in the C. difficile T4P and in interactions between the T4P and the host. Additionally they offer an exciting new target for a C. difficile vaccine.