Genome scanning for virulence genes in bacteria

描述(申请人提供)：在过去的五十年里，人类细菌感染的控制和治疗在很大程度上依赖于抗生素的使用。然而，耐药细菌的发病率不断增加，这就要求开发新的抗生素。全面了解对体外生长和体内生存至关重要的细菌基因，对于确定有效抗生素开发的有希望的靶点至关重要。该项目的目标是以肠炎链球菌(S.enteritidis)为模式生物，开发全基因组鉴定细菌基因的实验方法，这些基因对体外生长或体内生存至关重要。肠炎沙门氏菌是在美国引起食源性疾病的最常见的沙门氏菌血清型之一，它经常污染鸡蛋，对公共安全构成了独特的关注。我们最近开发了一种名为转座子序列标签分析(TSP)的方法，用于对复杂突变库中的转座子插入进行定量编目。通过对大约500株肠炎链球菌Tn5突变株的筛选，证明了TSP用于细菌基因组功能鉴定的可行性。为了实现这一目标，提出了两个具体目标。我们将使用基于TSP的实验方法对特定目的1的体外生长所必需的必需基因和在特定目的2的小鼠感染期间体内生存所需的毒力基因进行全基因组鉴定。本项目将鉴定的肠炎链霉菌的必需基因和毒力基因将为了解这种重要的食源性病原体的体外和体内存活机制提供深入的见解。这些实验方法将被广泛应用于其他不同的细菌物种，这将有助于开发有效的措施来控制细菌病原体。

DESCRIPTION (provided by applicant): The control and treatment of bacterial infections in humans have been largely dependent on the use of antibiotics for the last five decades. However, the increasing incidence of antibiotic resistant bacteria demands the development of novel antibiotics. Comprehensive knowledge of the bacterial genes that are essential for in vitro growth and in vivo survival is crucial to identify promising targets for the development of effective antibiotics. The goal of this project is to develop experimental approaches for genome-wide identification of bacterial genes that are essential for in vitro growth or in vivo survival using S. enterica serovar Enteritidis (S. enteritidis) as a model organism. S. enteritidis is one of the most common serotypes of Salmonella causing food borne illness in the United States, posing a unique concern for public safety by routinely contaminating chicken shell eggs. We recently developed a method, termed "transposon sequence tag profiling" (TSP), for quantitative cataloguing of transposon insertions in a complex pool of mutants. The feasibility of TSP in functional characterization of a bacterial genome was demonstrated by screening a pool of approximately 500 S. enteritidis Tn5 mutants for attenuation during chicken infection. Two specific aims are proposed to accomplish the goal. We will use experimental approaches based on TSP to conduct genome-wide identification of the essential genes that are not dispensable for in vitro growth in Specific Aim 1 and the virulence genes that are required for in vivo survival during mouse infection in Specific Aim 2. The essential genes and the virulence genes in S. enteritidis that will be identified in this project should provide insights into the mechanisms of both in vitro and in vivo survival of this important food borne pathogen. These experimental approaches should find broad applications to other diverse bacterial species, which will contribute to the development of effective measures to control bacterial pathogens.