Molecular analysis of Salmonella typhi host specificity

肠道沙门氏菌可引起人类和动物的多种疾病。伤寒沙门氏菌是伤寒的病原体，对其致病机制的了解受到实验动物缺乏毒力的限制。伤寒是一种宿主适应的血清型，专用于人类。我们的长期目标是更好地了解沙门氏菌导致人类感染和疾病的分子机制。我们的研究重点是通过广泛使用人类和小鼠细胞培养模型来了解伤寒杆菌的宿主特异性，以阐明这两个不同宿主物种在细菌基因表达和细胞内存活的差异。具体地说，我们将通过：(1)鉴定伤寒杆菌基因组中不存在的巨噬细胞中表达的伤寒杆菌特异基因，以及(2)鉴定仅在一种宿主细胞中特异表达的伤寒杆菌基因，以建立伤寒杆菌宿主特异性的遗传基础。在这些研究中，我们将使用新开发的分子遗传学工具，例如选择性捕获转录序列(SCOTS)，这是我们的研究小组目前使用的一种技术，用于识别在体内表达的细菌基因。我们还将使用一种独创的方法，使用GFP(绿色荧光蛋白)基因融合和细胞荧光测定法(FACS)。该项目将涉及使用自杀载体的定义缺失突变来产生突变菌株，并使用微生物遗传学、分子生物学、生物化学、免疫学、细胞生物学和显微镜的广泛方法。预计本申请中提出的研究将有助于我们了解伤寒杆菌是如何定植、入侵和克服宿主防御并引起肠热病的。这些信息应导致制定战略，包括开发疫苗以减少沙门氏菌定植和感染的可能性。

Salmonella enterica causes of a large variety of diseases in humans and animals. Understanding pathogenic mechanisms of S. enterica serovar Typhi, the etiologic agent of typhoid fever, is limited by lack of virulence in laboratory animals. Typhi is a host-adapted serovar that is specific for humans. Our long-term objective is to better understand the molecular mechanisms by which Salmonella causes infection and disease in humans. We will focus our research on understanding Typhi host specificity with extensive use of human and murine cell culture models to elucidate differences in bacterial gene expression and survival within cells from these two different host species. Specifically, we will conduct studies to establish a genetic basis for the host specificity of Typhi by : (1) characterizing Typhi specific genes expressed in macrophages that are absent from the serovar Typhimurium genome and (2) identification of Typhi genes that are specifically expressed in only one type of host cells. In these studies, we will employ newly developed molecular genetic tools, such as selective capture of transcribed sequences (SCOTS) a technique currently in use by our research group, that identifies bacterial genes expressed in vivo. We will also use an original approach using GFP (green fluorescent protein) gene fusions and cytofluorometry (FACS). This project will involve the generation of mutant strains using defined deletion mutations with suicide vectors and the use of a broad range of methods of microbial genetics, molecular biology, biochemistry, immunology, cell biology, and microscopy. It is anticipated that the research proposed in this application will contribute to our understanding of the means by which Typhi colonizes, invades and overcomes host defenses and causes enteric fever. This information should lead to strategies, including the development of vaccines to reduce likelihood of Salmonella colonization and infection.