Genome-wide identification of virulence genes in Acinetobacter baumannii in vivo

描述（由申请方提供）：鲍曼不动杆菌是一种革兰氏阴性细菌，已成为一种重要的机会致病菌，在美国构成重大公共卫生风险。临床上，A.鲍曼不动杆菌正在成为主要的医院病原体，特别是在专门从事呼吸护理、创伤和烧伤的重症监护病房中。尽管有三种A.在鲍曼不动杆菌中，哺乳动物宿主中致病所必需的大多数基因的身份是未知的。我们建议确定近4000个细菌基因中的哪些是在哺乳动物宿主中生存所必需的，并证明实验诱导菌血症期间每个基因的必要性。这一结果将代表着在理解A.鲍曼不动杆菌感染和设计策略来治疗这些感染或针对其发生进行疫苗接种。我们的长期目标是系统地建立A.鲍曼不动杆菌感染本申请的目的是鉴定所有基因 对在哺乳动物宿主内建立感染至关重要。在对109，000株A.鲍曼不动杆菌ATCC 17978在小鼠菌血症模型中的表达，我们的中心假设是A.可通过将全转座子诱变、鼠感染模型和高通量测序相结合来确定菌血症发展所必需的鲍曼不动杆菌。我们进行这项工作的基本原理是建立一个框架，将指导详细的机制调查A。鲍曼不动杆菌感染的发病机制利用A.本论文的主要目的是：1）鉴定鲍曼不动杆菌的致病基因;鲍曼不动杆菌在哺乳动物宿主内定殖和传播;和2）证实A.鲍曼不动杆菌毒力基因，其使用确定的突变在鼠模型中对定殖和传播贡献最显著。这一贡献将是重大的，因为在这些全面且无偏见的实验中，我们将首次确定完整的A。在哺乳动物感染期间两种菌株的鲍曼不动杆菌毒力基因集。这一建议应被视为创新，因为它适用于转座子定向插入位点测序（TraDIS）的全基因组鉴定的A。在相关的感染动物模型中使用正向遗传筛选的鲍曼不动杆菌毒力基因。

DESCRIPTION (provided by applicant): Acinetobacter baumannii, a Gram-negative bacterium, has emerged as an important opportunistic pathogen that poses a significant public health risk in the United States. Clinically, A. baumannii is emerging as a leading nosocomial pathogen, particularly in intensive care units specializing in respiratory care, trauma and burns. Despite having the complete genome sequences for three strains of A. baumannii, the identity of most genes that are essential for pathogenesis in a mammalian host is not known. We propose to identify which of the nearly 4000 bacterial genes are essential for survival in a mammalian host and to demonstrate the necessity for each gene during experimentally induced bacteremia. The result will represent a major step forward in understanding the pathobiology of A. baumannii infections and for devising strategies to treat these infections or vaccinate against their occurrence. Our long-term goal is to systematically establish a molecular model of pathogenesis for A. baumannii infection. The objective of this application is to identify all genes essential to establish infection within a mammalian host. Based on a successful pilot experiment with 109,000 mutant of A. baumannii ATCC17978 in a murine model of bacteremia, our central hypothesis is that genes of A. baumannii essential for development of bacteremia can be determined by combining global transposon mutagenesis, a murine model of infection, and high throughput sequencing. Our rationale for conducting this work is to establish a framework that will direct detailed mechanistic investigations of A. baumannii pathogenesis in compromised patients. Using a saturating transposon library of A. baumannii mutants, the animal model of bacteremia, and high- throughput DNA sequencing, we will accomplish the following specific aims: 1) identify virulence genes essential for A. baumannii to colonize and disseminate within a mammalian host; and 2) confirm A. baumannii virulence genes that contribute most significantly to colonization and dissemination in the murine model using defined mutations. This contribution will be significant because, in these comprehensive and unbiased experiments, we will determine for the first time the complete A. baumannii virulence gene set for two strains during a mammalian infection. This proposal should be considered innovative as it applies transposon-directed insertion-site sequencing (TraDIS) for genome-wide identification of A. baumannii virulence genes using a forward-genetic screen in a relevant animal model of infection.