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

描述(申请人提供)：鲍曼不动杆菌，一种革兰氏阴性细菌，已成为一种重要的机会致病菌，在美国构成重大的公共健康风险。临床上，鲍曼不动杆菌正在成为一种主要的医院病原体，特别是在呼吸护理、创伤和烧伤的重症监护病房。尽管拥有鲍曼不动杆菌三个菌株的完整基因组序列，但在哺乳动物宿主中致病所必需的大多数基因的同一性尚不清楚。我们建议确定近4000个细菌基因中哪些是在哺乳动物宿主中生存所必需的，并证明每个基因在实验诱导的菌血症中的必要性。这一结果将是在了解鲍曼不动杆菌感染的病理生物学以及设计治疗这些感染或接种疫苗预防它们发生的策略方面向前迈出的重要一步。我们的长期目标是系统地建立鲍曼不动杆菌感染发病机制的分子模型。这个应用程序的目标是识别所有基因 在哺乳动物宿主内建立感染所必需的。基于在小鼠菌血症模型中对109,000个鲍曼不动杆菌ATCC17978突变体进行的成功的初步实验，我们的中心假设是，通过结合全球转座子突变、小鼠感染模型和高通量测序，可以确定鲍曼不动杆菌发生菌血症所必需的基因。我们进行这项工作的基本原理是建立一个框架，指导对鲍曼不动杆菌致病机制的详细研究。利用饱和的鲍曼不动杆菌突变体转座子文库、菌血症动物模型和高通量DNA测序，我们将实现以下特定目标：1)确定鲍曼不动杆菌在哺乳动物宿主内定植和传播所必需的毒力基因；2)利用已定义的突变，确认对鲍曼不动杆菌在小鼠模型中定植和传播最重要的毒力基因。这一贡献将是重大的，因为在这些全面和公正的实验中，我们将首次确定哺乳动物感染期间两个菌株的完整鲍曼不动杆菌毒力基因集。这一建议应该被认为是创新的，因为它应用了转座子定向插入位点测序(TRADIS)来在相关动物感染模型中使用正向遗传筛选来识别鲍曼不动杆菌毒力基因的全基因组。

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.