Regulation of stress resistance and virulence genes in Acinetobacter baumannii

 描述(由申请人提供)：鲍曼不动杆菌是一种严格需氧的革兰氏阴性球菌，最近作为一种重要的机会人类病原体出现。这种细菌最常与医院感染有关，通常被认为是一种机会性的人类病原体，尽管有一些社区获得性感染的报道。鲍曼不动杆菌引起的疾病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。鲍曼不动杆菌感染因多药耐药(MDR)菌株的增加而进一步复杂化。目前，人们对鲍曼不动杆菌的致病机制知之甚少。目前提出的毒力决定因素包括铁载体的产生、衣壳多糖的精制、生物膜的产生、细菌外膜蛋白A(OmpA)以及其他几种功能未知的候选蛋白。关于毒力相关基因是如何调控的信息就更少了。为了发现鲍曼不动杆菌的毒力基因，我们利用了一种无脊椎动物昆虫模型，该模型已被用于研究各种感染源，如蜡蛾的幼虫。我们使用鲍曼不动杆菌多药耐药致病株AB5075基因组中的大量转座子插入突变进行了正向遗传筛查。通过对细菌中转座子插入位点的测序，我们鉴定了180多个基因，这些基因似乎是幼虫生存所特需的，对在富媒体中的生存和生长是必不可少的。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个基因被预测为编码与抗病相关的蛋白 存在渗透胁迫。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。这些突变株在幼虫血淋巴中的存活率也比野生型鲍曼不动杆菌AB5075低。这项拟议的工作包括构建28个基因(17个调节基因和11个抗逆性基因)的缺失突变，并在Galleria模型中测试它们的表型。将评估相应的野生型基因对表现出表型的突变体的补充能力。RNAseq的转录图谱将在Galeria幼虫模型中显示毒力降低的几个突变体上进行。这项工作将对一种新出现的人类病原体的毒力的遗传基础产生重要的新知识。

 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.