Rewiring networks for a pathogenic lifestyle

项目摘要 霍乱弧菌引起严重的霍乱，是亚洲、非洲和南 最近在海地、叙利亚和乌克兰重新出现。物种多样性很高，但只有 O 1或O 139血清群菌株引起流行性疾病。然而，越来越多的散发性疾病 全球报告，由属于非O 1/非O 139血清群的菌株引起， 健康威胁在发达国家和工业化国家，包括美国。与致病性O 1不同 和O 139菌株，绝大多数致病性非O 1/非O 139菌株不携带良好表征的 定殖毒力因子（TCP）和毒素产生毒力因子（CT），以及这些毒力因子使用的毒力机制。 菌株还不太清楚。我们对致病性非O 1/非O 139血清群菌株的研究始于 临床分离的O39血清群菌株AM-19226的基因组测序，其揭示了三型 分泌系统（T3 SS）是其他霍乱弧菌分离株中保守的。与TCP和CT一样，T3 SS 通过水平基因转移（HGT）获得，并整合到祖先染色体中。我们随后 实验鉴定了T3 SS中编码的两种膜定位转录激活因子（MLTA 基因组致病岛（派）是T3 SS在体内和体外发挥功能所必需的。我们还发现 ToxR是TCP和CT表达所需的祖先MLTA，对T3 SS调节很重要。ToxR是 由霍乱弧菌的所有菌株编码，在那里它调节核心染色体功能，并且作为一种 MLTA交叉调节派和祖先基因表达。有趣的是，我们发现T3 SS编码了 MLTA影响祖先基因表达和表型，如运动性和生物膜形成。我们 假设为了在水生环境中生存并引起疾病，T3 SS阳性霍乱弧菌 必须将毒力基因调控与派外的转录回路整合， 无论是祖先的和新获得的，派编码的转录调节因子。我们建议使用 互补的遗传和生物化学方法，以确定和表征T3 SS使用的机制 PAI协调运动，生物膜形成，T3 SS基因表达对环境的提示。 初步研究将确定T3 SS MLTA的调节子，以及促进调节的条件。我们将 研究蛋白质-蛋白质相互作用和MLTA转录结构域序列特异性。初步研究将 关注VttRA作为处于调控层级顶端的蛋白质。我们的总体目标是发现派 编码的MLTA活性调节毒力表型和祖先的生理特性， 维持新进化的病原体的双重生活方式。

Project Summary Vibrio cholerae causes the severe diarrheal disease cholera that is endemic in much of Asia, Africa, and South America, and has recently reemerged in Haiti, Syria, and Ukraine. The species is highly diverse, although only O1 or O139 serogroup strains cause epidemic disease. However, increasing sporadic disease has been reported globally, and is caused by strains belonging to non-O1/non-O139 serogroups that present a public health threat both in developed and industrialized nations, including the United States. 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 the virulence mechanisms used by these strains are not well understood. Our study of pathogenic non-O1/non-O139 serogroup strains began with genomic sequencing of the clinically isolated O39 serogroup strain, AM-19226, which revealed a Type Three Secretion System (T3SS) that is conserved among other V. cholerae isolates. Like TCP and CT, the T3SS is acquired by horizontal gene transfer (HGT), and integrated into the ancestral chromosome. Our subsequent experiments identified two membrane localized transcriptional activators (MLTAs) encoded within the T3SS genomic pathogenicity island (PAI), which are essential for T3SS function in vivo and in vitro. We also found that ToxR, an ancestral MLTA required for TCP and CT expression, is important for T3SS regulation. ToxR is encoded by all strains of V. cholerae where it regulates core chromosomal functions, and is well-studied as an MLTA that cross-regulates PAI and ancestral gene expression. Interestingly, we found that the T3SS encoded MLTAs influence ancestral gene expression and phenotypes such as motility and biofilm formation. We hypothesize that in order to survive in aquatic environments and also cause disease, T3SS-positive V. cholerae must integrate virulence gene regulation with transcriptional circuits outside of the PAI, leveraging the activities of both ancestral and newly acquired, PAI encoded transcriptional regulatory factors. We propose to use complementary genetic and biochemical approaches to identify and characterize the mechanisms used by T3SS PAIs to coordinate motility, biofilm formation, and T3SS gene expression in response to environmental cues. Initial studies will define the regulons for T3SS MLTAs, and the conditions promoting regulation. We will investigate protein-protein interactions and MLTA transcriptional domain sequence specificity. Initial studies will focus on VttRA as the protein at the top of the regulatory hierarchy. Our overall goal is to discover how PAI encoded MLTA activity regulates virulence phenotypes and ancestral physiological traits that are necessary to maintain dual lifestyles of newly evolved pathogens.