Molecular Pathogenesis of Vibrio vulnificus

创伤弧菌的分子发病机制

• 批准号: 6779075
• 负责人: PAUL A GULIG
• 金额: $ 29.04万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779075
• 关键词: Molecular; Pathogenesis; Vibrio; vulnificus

DESCRIPTION (provided by applicant): Vibrio vulnificus is a gram-negative bacterium that causes fulminating diseases in susceptible humans: septicemia after ingestion of oysters and wound infection from seawater. The major predisposing factor is iron overload. The mortality rates for septicemia and wound infection are 77% and 15%, respectively, and patients can die within 24 hours of contact with the bacteria. V. vulnificus is highly invasive and replicates rapidly in host tissues, leading to high numbers of bacteria and extensive tissue damage. Our use of subcutaneously inoculated, iron dextran-treated mice revealed extensive tissue damage that resembles human disease and differentiated virulent clinical strains from less virulent oyster strains. Virulent V. vulnificus replicated extremely rapidly in the mice and were resistant to PMNs. Our hypothesis is that the rapid growth rate of V. vulnificus in host tissues and resistance to host phagocytic defenses enable the bacteria to reach high numbers and cause tissue damage with multifactorial toxins. Our preliminary results have enabled the dissection of each of these factors in the animal model using genetic tools in use by us. We propose to continue to use a molecular genetic approach to identify virulence factors of V. vulnificus. The specific aims are to: 1) Use a combination of signature-tagged mutagenesis (STM), PhoA fusion/insertion mutagenesis, and in vivo selection for complementation of naturally occurring attenuating mutations to identify virulence genes of V. vulnificus, 2) Use a marker plasmid with the iron dextran-treated mouse model to differentiate the effects of virulence genes on growth, killing, and damage, and use in vitro models to characterize the virulence phenotypes in more detail, and 3) Complete the molecular version of Koch's postulates for important virulence genes. In aim 1 we will primarily use STM to obtain mutations in genes involved with rapid growth or evasion of PMNs. We will use PhoA fusion/insertion mutagenesis to identify genes encoding secreted proteins. In aim 2 after differentiating effects of mutations on growth in versus killing by the host, we will characterize damage by examining histopathology and examine the interaction of vibrios with PMNs by infecting neutropenic mice. In vitro assays will involve analysis of auxotrophy, iron acquisition, complement resistance, PMN resistance, and cytotoxicity to cell culture. These studies will elucidate mechanisms of fulminating, invasive disease caused by V. vulnificus as related to rapid replication, evasion of defenses, and damage to host tissues.

描述（由申请人提供）：创伤弧菌是一种革兰氏阴性细菌，在易感人群中引起暴发性疾病：摄入牡蛎后败血症和海水伤口感染。主要的诱发因素是铁超载。败血症和伤口感染的死亡率分别为77%和15%，患者可能在接触细菌后24小时内死亡。创伤弧菌具有高度侵袭性，并在宿主组织中快速复制，导致大量细菌和广泛的组织损伤。我们使用皮下接种，铁葡聚糖处理的小鼠发现广泛的组织损伤，类似于人类疾病和区分毒性临床菌株从毒性较低的牡蛎菌株。毒性创伤弧菌在小鼠中复制非常迅速，并对中性粒细胞具有抗性。我们的假设是，创伤弧菌在宿主组织中的快速生长速度和对宿主吞噬防御的抵抗力使细菌能够达到高数量，并通过多因子毒素引起组织损伤。我们的初步结果使我们能够使用遗传工具在动物模型中解剖这些因素中的每一个。 我们建议继续使用分子遗传学方法来鉴定创伤弧菌的毒力因子。具体目标是：1）使用特征标记诱变（STM）、PhoA融合/插入诱变和用于天然存在的减毒突变的互补的体内选择的组合来鉴定创伤弧菌的毒力基因，2）使用标记质粒与铁葡聚糖处理的小鼠模型来区分毒力基因对生长、杀伤和损伤的影响，并利用体外模型更详细地描述毒力表型; 3）完成重要毒力基因的Koch假设的分子版本。在目标1中，我们将主要使用STM来获得与PMN的快速生长或逃避有关的基因突变。我们将使用PhoA融合/插入诱变来鉴定编码分泌蛋白的基因。在目标2中，在区分突变对宿主生长与杀伤的影响后，我们将通过检查组织病理学来表征损伤，并通过感染贫血小鼠来检查弧菌与PMN的相互作用。体外试验将涉及营养缺陷型、铁获得、补体抗性、PMN抗性和对细胞培养物的细胞毒性的分析。 这些研究将阐明创伤弧菌引起的暴发性、侵袭性疾病的机制，这些机制与快速复制、逃避防御和对宿主组织的损伤有关。