Iron and pathogenesis in infections by Vibrio vulnificus

描述(由申请人提供)：创伤弧菌是一种机会性人类病原体，能够导致致命的原发败血症或坏死性伤口感染。败血症发生在免疫功能受损或患有血色素沉着症或其他潜在肝脏疾病的患者，如肝硬变和酒精性肝病。大多数患者的共同主题是铁的存在高于生理水平。我们认为创伤弧菌CMCP6的基因调控取决于许多因素，包括感染过程中高铁、营养物质浓度和氧气供应等环境条件的变化。在这一应用中，我们建议剖析它们在创伤弧菌体内和体外表达的具体机制。实现这些目标的具体目标是：1)在体外调节受感染患者血清中铁浓度和/或人血清成分影响的基因的表达。为了达到这个目的，我们发现除了血清中的高铁浓度外，还有一些因素对患者血清中表达的毒力基因的表达也有影响。2)创伤弧菌在高铁限制条件下表达的转录调控因子和毒力因子HlyU的分析。我们证明，HlyU除了是一个重要的毒力因子外，还是一个全球转录调控因子，在富铁和限铁条件下都控制着许多毒力基因的表达，其中一些基因只有在高铁条件下才高表达。为此，我们建议对纯化的HlyU蛋白进行转录和翻译融合以及凝胶移动和DNAsel保护实验。3)创伤弧菌基因在两种不同小鼠模型中的体内表达分析。为此，我们建议使用基于体内重组的技术(RIVET)来识别创伤弧菌在铁过载小鼠模型感染过程中体内特异表达的基因，并与感染正常小鼠后诱导的基因进行比较。使用两种小鼠模型以及在体外鉴定的基因将通过突变和毒力实验来表征。因此，我们希望与体外生长相比，获得这种细菌在体内的生理学的全面图像。这些研究将有助于加深对创伤弧菌感染的发病机制以及一般细菌毒力的了解。

DESCRIPTION (provided by applicant): Vibrio vulnificus is an opportunistic human pathogen capable of causing fatal primary septicemias or necrotizing wound infections. Septicemia occurs in patients that are immunocompromised or suffering from hemochromatosis or with other underlying liver disorders such as cirrhosis and alcoholic liver disease. The common theme in most of these patients is that iron is present at higher than physiological level. We believe that gene regulation of V. vulnificus CMCP6 depends on many factors according to the changes in environmental conditions i.e. high iron, nutrients concentration, and oxygen availability during the process of infection. In this application we propose to dissect the specific mechanisms that govern their expression in V. vulnificus, in vitro and in vivo. The specific aims to achieve these goals are: 1) In vitro regulation of genes whose expression is affected by the iron concentration of the medium and/or components of human serum of compromised patients. In this aim we identify factors in addition to the high iron concentration of the serum play a role in the expression of virulence genes expressed in patient sera. 2) Analysis of HlyU, a global transcription regulator and virulence factor of V. vulnificus expressed at both high and iron limiting conditions. We demonstrate that HlyU in addition to being an important virulence factor is also a global transcriptional regulator that at both iron-rich and iron limiting conditions controls the expression of many virulence-genes, some of them highly expressed only at high iron conditions. In this aim we propose to use a combination of transcriptional and translational fusions as well as gel shift and DNAsel protection experiments with the purified HlyU protein. 3) Analysis of the in vivo expression of V. vulnificus genes in two different mouse models. In this specific aim we propose to use recombination-based in vivo technology (RIVET) to identify V. vulnificus genes that are expressed specifically in vivo during infection of the iron-overloaded mouse model as compared to those induced after infecting the normal mouse. The genes identified using both mouse models as well as those identified in vitro will be characterized by mutagenesis and virulence experiments. Thus, we expect to obtain a comprehensive picture of the physiology of this bacterium during in vivo as compared to in vitro growth. These studies will lead to an enhanced understanding of the pathogenesis of V. vulnificus infections in particular and of bacterial virulence in general.