Genome scanning for virulence genes in bacteria

描述（由申请人提供）：在过去的50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，抗生素耐药菌的不断增加要求开发新型抗生素。全面了解细菌基因对体外生长和体内生存至关重要，这对于确定有效抗生素的有希望的靶点至关重要。该项目的目标是开发全基因组鉴定细菌基因的实验方法，这些基因是体外生长或体内生存所必需的，以肠球菌血清型肠炎（S. enterititis）为模式生物。肠炎沙门氏菌是在美国引起食源性疾病的沙门氏菌最常见的血清型之一，通过常规污染鸡壳蛋，对公共安全提出了独特的关注。我们最近开发了一种称为“转座子序列标记分析”（TSP）的方法，用于在复杂的突变体池中对转座子插入进行定量编目。通过筛选大约500个肠炎沙门氏菌Tn5突变体，在鸡感染期间进行衰减，证明了TSP在细菌基因组功能表征中的可行性。为实现这一目标，提出了两个具体目标。我们将使用基于TSP的实验方法，对特异性Aim 1中体外生长不需要的必需基因和特异性Aim 2中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。本项目将确定肠炎沙门氏菌的必需基因和毒力基因，为了解这一重要食源性病原体的体内和体外生存机制提供依据。这些实验方法应该在其他不同的细菌种类中找到广泛的应用，这将有助于开发有效的控制细菌病原体的措施。

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.