Molecular Pathogenesis of Vibrio vulnificus

描述（由申请人提供）：创伤弧菌是一种革兰氏阴性菌，可在易感人群中引起严重疾病：食用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%，患者可在接触细菌24小时内死亡。创伤弧菌具有很强的侵袭性，在宿主组织中复制迅速，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显示了广泛的组织损伤，类似于人类疾病，并区分了强毒的临床菌株和弱毒的牡蛎菌株。毒力极强的创伤弧菌在小鼠体内复制极快，并对PMNs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其数量达到很高，并通过多因子毒素造成组织损伤。我们的初步结果使我们能够使用遗传工具对动物模型中的每个因素进行解剖。

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.