Pathogenic mechanisms of non-01/non-0139 V. cholerae

描述（由申请人提供）：霍乱弧菌是严重霍乱的病原体，霍乱是亚洲、非洲和南美洲大部分地区的地方病。虽然只有O 1或O 139血清群的菌株会引起流行病，但该物种是相当多样化的。然而，大量的疾病在全球范围内以零星发作的形式发生，并且是由属于非O 1/非O 139血清群的菌株引起的。与致病性O 1和O 139菌株不同，绝大多数致病性非O 1/非O 139菌株不携带已充分表征的定植毒力因子（TCP）和毒素产生因子（CT），并可能通过未知机制引起疾病。临床分离的非O 1/非O 139菌株AM-19226的全基因组测序揭示了开放阅读框（ORF）的存在，其与编码三型分泌系统（TTSS）的基因具有显著的相似性。这些ORF似乎在非O 1/非O 139菌株的子集以及副溶血性弧菌的大流行菌株中是保守的。许多病原菌使用TTSS将毒力因子转移到宿主细胞的胞质溶胶中，我们假设霍乱弧菌中存在类似的ORF代表了这些菌株获得的宿主细胞相互作用和毒力的新鉴定机制。实验表明，TTSS确实是功能性的，并在发病机制中发挥作用。TTSS关键组分的缺失严重减弱了菌株AM-19226在幼鼠模型中定殖的能力。此外，已经鉴定了一种效应蛋白（其分泌依赖于TTSS），并且它似乎在宿主细胞肌动蛋白的重组中起作用。我们建议使用三种互补的体外方法来开始了解TTSS介导的霍乱弧菌发病机制。第一种方法是使用互补的体外和体内模型系统来鉴定促进毒力的其他效应蛋白。第二种方法是剖析效应蛋白如何在分子水平上与宿主细胞相互作用而导致疾病，第三种方法是使用基因组方法来开始理解控制编码效应蛋白的基因表达的调控网络和TTSS结构装置。项目叙述：今天，霍乱被认为是一种健康威胁，主要发生在发展中国家、缺乏现代卫生设施的地区以及因战争或环境危机而遭受民用基础设施破坏的国家。这种疾病对前往流行地区的个人构成威胁，美国疾病控制和预防中心认为一些菌株属于B级生物恐怖主义威胁，因为它们有可能通过受污染的食物或水传播。虽然O 1和O 139引起流行病的菌株得到了很好的研究，但本提案旨在了解代表新兴威胁的其他血清群菌株所采用的毒力机制。

DESCRIPTION (provided by applicant): Vibrio cholerae is the causative agent of the severe diarrheal disease, cholera, that is endemic in much of Asia, Africa, and South America. The species is quite diverse, although only strains of the O1 or O139 serogroup cause epidemic disease. However, a significant amount of disease occurs globally in sporadic episodes and is caused by strains belonging to non-O1/non-O139 serogroups. Unlike pathogenic O1 and O139 strains, the vast majority of pathogenic non-O1/non-O139 strains do not carry the well characterized virulence factors for colonization (TCP) and toxin production (CT), and presumably cause disease by an unknown mechanism(s). Whole genome sequencing of a clinically isolated non-O1/non-O139 strain, AM-19226, has revealed the presence of open reading frames (ORFs) having significant similarity to genes encoding a Type Three Secretion System (TTSS). These ORFs appear to be conserved among a subset of non-O1/non-O139 strains, as well as pandemic strains of V. parahaemolyticus. Many pathogenic bacteria use TTSSs to translocate virulence factors into the cytosol of host cells, and we hypothesize that the presence of similar ORFs in V. cholerae represents a newly identified mechanism for host cell interaction and virulence acquired by these strains. Experiments suggest that the TTSS is indeed functional and has a role in pathogenesis. Deletion of a critical component of the TTSS severely attenuates the ability of strain AM-19226 to colonize the infant mouse model. Furthermore, an effector protein (whose secretion is TTSS dependent) has been identified, and it appears to have a role in the reorganization of host cell actin. We propose to use three complimentary in vitro approaches to begin to understand TTSS mediated pathogenesis in V. cholerae. The first approach is to identify additional effector proteins that promote virulence, using complimentary in vitro and in vivo model systems. The second approach is to dissect how effector proteins interact with host cells at the molecular level to cause disease, and the third is to use genomic approaches to begin to understand the regulatory network governing expression of the genes encoding effector proteins and the TTSS structural apparatus. Project Narrative: Today cholera is considered a health threat mainly in developing nations, regions lacking modern sanitation facilities, and in countries experiencing disrupted civil infrastructure due to war or environmental crises. The disease poses a threat to individuals traveling to endemic areas, and the United States CDC considers some strains a Category B level BioTerrorism threat because of the potential for spread via contaminated food or water. While O1 and O139 epidemic causing strains are well studied, this proposal seeks to understand the virulence mechanisms employed by strains of other serogroups that represent an emerging threat.