Regulation of stress resistance and virulence genes in Acinetobacter baumannii

 描述（由申请方提供）：鲍曼不动杆菌是一种严格需氧的革兰氏阴性球杆菌，最近成为一种重要的人类机会致病菌。这种微生物最常与医院感染相关，通常被认为是一种机会性人类病原体，尽管有一些社区获得性感染的报道。A.引起的疾病鲍曼不动杆菌的感染包括皮肤和软组织感染、尿路感染以及更具侵袭性的疾病，如心内膜炎、败血症、骨髓炎和肺炎。A.鲍曼不动杆菌感染由于多药耐药（MDR）菌株的流行增加而进一步复杂化。目前，对A.鲍曼不动杆菌毒力机制目前提出的毒力决定因素包括铁载体的产生，荚膜多糖的加工，生物膜的产生，细菌外膜蛋白A（OmpA），以及其他几个未知功能的候选蛋白。关于毒力相关基因是如何调控的信息甚至更少。为了发现A.为了研究鲍曼不动杆菌的毒力基因，我们利用了一种无脊椎昆虫模型，该模型已被用于研究各种感染因子，蜡螟的幼虫，大蜡螟。我们利用一个MDR致病株的基因组中大量的转座子插入突变进行了正向遗传筛选。鲍曼不动杆菌，AB5075。通过对在富培养基中生长的细菌和感染的幼虫中的转座子插入位点进行测序，我们确定了180多个基因，这些基因似乎是幼虫生存和在富培养基中生存和生长所必需的。其中17个基因编码的产物被注释为DNA结合蛋白或其他类型的调节蛋白。此外，11个基因预测编码与抗性相关的蛋白质， 我们创建了注释为海藻糖合成酶（otsB）、钾通道（trkAH）和机械敏感通道（mscS）的基因的缺失。与野生型A相比，这些突变体在幼虫血淋巴中的存活率也降低。鲍曼不动杆菌AB 5075。这项工作包括构建28个基因（17个调节基因和11个抗逆基因）的缺失突变，并在Galleria模型中测试它们的表型。将评价相应野生型基因与表现出表型的突变体互补的能力。将对在Galleria幼虫模型中表现出毒力降低的几种突变体进行RNAseq转录谱分析。这项工作将导致在一个新兴的人类病原体的毒力的遗传基础的重要的新知识。

 DESCRIPTION (provided by applicant): Acinetobacter baumannii is a strictly aerobic, gram-negative coccobacillus that has recently emerged as a significant opportunistic human pathogen. This organism is most commonly associated with nosocomial infections and is generally considered to be an opportunistic human pathogen, although some community acquired infections are reported. Diseases caused by A. baumannii include skin and soft tissue infections, infections of the urinary tract, as well as more invasive diseases such as endocarditis, sepsis, osteomyelitis and pneumonia. A. baumannii infections are further complicated by the increasing prevalence of multidrug resistant (MDR) strains. At present, little is known about A. baumannii virulence mechanisms. Currently proposed virulence determinants include the production of siderophores, elaboration of capsular polysaccharides, biofilm production, the bacterial outer membrane protein A (OmpA), as well as several other candidate proteins of unknown function. There is even less information about how virulence-related genes are regulated. To discover A. baumannii virulence genes, we took advantage of an invertebrate insect model that has been used to study a variety of infectious agents, the larva of the wax moth, Galleria mellonella. We conducted a forward genetic screen using a large pool of transposon insertion mutations in the genome of a MDR pathogenic strain of A. baumannii, AB5075. By sequencing the insertion sites of the transposons in bacteria grown in rich media and from infected larvae, we identified more than 180 genes that appear to be specifically required for survival in larvae and dispensable for survival and growth in rich media. Seventeen of the genes encode products that are annotated as either DNA binding proteins or other types of regulatory proteins. In addition, 11 genes predicted to encode proteins related to resistance to osmotic stress were found. We created deletions of the genes annotated as trehalose synthetase (otsB), potassium channels (trkAH), and a mechanosensitive channel (mscS). These mutants also exhibited decreased survival in larva hemolymph compared to wild-type A. baumannii AB5075. This proposed work includes construction of deletion mutations of the 28 genes (17 regulators and 11 stress resistance) and testing their phenotypes in the Galleria model. The ability of the corresponding wild-type genes to complement mutants that exhibit a phenotype will be evaluated. Transcriptional profiling by RNAseq will be performed on several mutants that exhibit decreased virulence in the Galleria larva model. This work will result in important new knowledge about the genetic basis of virulence in an emerging human pathogen.