Molecular Pathogenesis of Vibrio vulnificus

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： 进食牡蛎后的败血症和海水造成的伤口感染。主要诱发因素是铁 超载。败血症和伤口感染的死亡率分别为77%和15%， 在接触细菌后24小时内死亡。创伤弧菌具有高度侵袭性，在宿主体内复制迅速 组织，导致大量的细菌和广泛的组织损伤。我们使用皮下接种， 铁葡聚糖治疗的小鼠显示出类似于人类疾病的广泛组织损伤， 毒性较低的牡蛎菌株的毒性临床菌株。致命的创伤弧菌在 小鼠和耐中性粒细胞。我们的假设是，创伤弧菌在宿主体内的快速生长速度 组织和对宿主吞噬防御的抗性使细菌能够达到高数量， 多因子毒素造成的组织损伤我们的初步研究结果使我们能够解剖出 使用我们使用的遗传工具在动物模型中的因素。 我们建议继续使用分子遗传学方法来鉴定Vu/nificus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，以及用于互补天然存在的 减毒突变以鉴定创伤弧菌的毒力基因，2）使用具有铁的标记质粒， 葡聚糖处理的小鼠模型，以区分毒力基因对生长、杀伤和 损伤，并使用体外模型来更详细地表征毒力表型，以及3）完成 重要毒力基因的科赫假设的分子版本。在aim 1中，我们将主要使用 STM以获得与PMN快速生长或逃避有关的基因突变。我们将使用PHOA 融合/插入诱变以鉴定编码分泌蛋白的基因。在目标2中，在区分 突变对生长的影响与宿主的杀伤作用，我们将通过检查组织病理学来表征损伤， 通过感染贫血小鼠检测弧菌与中性粒细胞的相互作用。体外试验将涉及以下分析： 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。 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， 快速复制、逃避防御和对宿主组织的损害。

Vibrio vulnificus is a gram-negative bacterium that causes fulminating diseases in susceptible humans: septicemia after ingestion of oysters and wound infection from sea water. 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. vu/nificus. 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.