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

艰难梭菌 IV 型菌毛亚基的功能和疫苗潜力

• 批准号: 8784829
• 负责人: GRACE ANNE MALDARELLI
• 金额: $ 3.32万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-29 至 2018-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8784829
• 关键词: Adherence; Adhesions; Adjuvant; Anaerobic Bacteria; Antibiotic Therapy; Antibiotics; Antibodies; Antibody Formation; Bacteria; Bacterial Adhesins; Bacterial Typing; Binding; Biogenesis; Caco-2 Cells; Caliber; Cell Line; Cell surface; Cells; Cessation of life; Characteristics; Clostridium difficile; Colectomy; Colon; Complement Receptor; Control Groups; DNA; Data; Diarrhea; Disease; Dissociation; Epithelial Cells; Event; Fimbriae Proteins; Flow Cytometry; Fluorescence Microscopy; Gene Components; Generations; Genome; Haploid Cells; Health; Hela Cells; Histopathology; Human; Immunization; Immunoblotting; Infection; Infection prevention; Inflammation; Intestines; Investigation; Lead; Measurement; Measures; Mediating; Meningitis; Minor; Modeling; Morbidity - disease rate; Mus; Oryctolagus cuniculus; Outcome; Pathogenesis; Patients; Physicians; Pilum; Preparation; Prevalence; Prevention; Proteins; Pseudomembranous Colitis; Recording of previous events; Relapse; Reproduction spores; Research; Role; Scientist; Structure; Surface; System; Testing; Therapeutic; Tissue Sample; Toxic Megacolon; Training; Transplantation; United States; Vaccines; Vibrio cholerae; Virulence; Work; appendage; cell motility; comparative efficacy; crosslink; efficacy testing; enteropathogenic Escherichia coli; extracellular; human APEX1 protein; improved; mortality; novel strategies; pathogen; protein structure; public health relevance; receptor; receptor expression; research study; response

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.

描述（由申请人提供）：艰难梭菌是一种革兰氏阳性，形成孢子的，强大的厌食症，已成为美国腹泻的主要原因。细菌感染具有多种表现，从结肠无症状到大量腹泻，假膜结肠炎和死亡。尽管治疗可能失败，并且患者可以复发，但仍可以接受感染的抗生素治疗。其他可用的疗法包括粪便移植以及在非常严重的结肠切除术。艰难梭菌感染每年14,000人死亡；因此，迫切需要更好地了解其发病机理以及改进的预防措施。尽管仍在研究初始定植中的确切事件序列，但Othr肠道病原体的证据表明，对上皮细胞的附着是发病机理的必要步骤。最近在艰难梭菌基因组中鉴定出用于粘附和运动性的一种细菌附属物的IV型菌毛（T4P）的基因。 T4P的主体由Pilins组成。主要的结构亚基是主要的PILIN，而其他类似结构的蛋白质称为小pilins或pilin样蛋白质，存在于较低的丰度，并且在Pilus Biogenesis中具有不同的作用。艰难梭菌基因组编码9个特定的pilin或pilin样蛋白。该提案着重于功能和疫苗电位 一种这样的蛋白质，pilj。我假设皮尔（Pilj）是次要的皮林（Pilin），并且比预测的pilin pila1 pila1的富含丰度低。我将使用定量免疫印迹和QRT-PCR来检验此假设。此外，我假设PILJ是T4P蛋白，并且它与宿主细胞上的特定受体相互作用。为了识别该受体，我将采用两种不同的方法。一种涉及将PILIN交联的受体，而另一个则利用人单倍体细胞系。最后，我假设在艰难梭菌感染的鼠模型中，对PILJ和辅助的免疫将防止艰难梭菌的挑战。 用PILJ的小鼠和兔子的免疫产生对蛋白质的强烈而特异性的反应。我预测这种反应将具有保护性。该实验的结果将是死亡率，发病率，组织病理学和末端抗体滴度。这些结果将阐明PILJ在艰难梭菌T4P中的作用以及T4P与宿主之间的相互作用。此外，它们为艰难梭菌疫苗提供了令人兴奋的新目标。