INVESTIGATING TYPE VI SECRETION IN ACINETOBACTER BAUMANNII AND ITS INTERPLAY WITH ANTIBIOTIC RESISTA

项目摘要/摘要 多药耐药鲍曼不动杆菌已成为一种常见的致病因素 院内感染，部分分离株对所有临床相关抗生素耐药。我们有 以前在这种生物中发现了一种VI型分泌系统(T6SS)。多组分 T6SS装置有助于在体内动态接触注射有毒效应蛋白 相互竞争的细菌。T6SS是能量昂贵的，因此在大多数细菌中似乎 受到严格的监管。我们最近发现，鲍曼不动杆菌的几个多药耐药株含有一种 一种大的、可自我传播的、负调控T6SS的抗性质粒。我们发现T6SS 在含有质粒的抗药性细胞中是沉默的，而部分种群 经常经历T6SS的质粒丢失和激活。这种激活导致了T6SS- 通过媒介杀死竞争细菌，但使鲍曼不动杆菌对抗生素敏感。我们 提出鲍曼不动杆菌细胞向细菌杀菌剂的分化涉及多个 表型和代谢变化以及与MDR和 T6SS表型是这种分化的驱动力。RNAseq与差异 定量蛋白质组学实验显示，意想不到的代谢途径与 氨基酸分解代谢受质粒调控。这些新陈代谢变化中的大多数似乎是 对T6SS激活和携带MDR质粒的能量适应的结果。通过 诱变和比较适合度分析我们将确定这些 代谢变化。干扰这些途径可能会产生对抗A. 鲍曼氏杆菌感染。我们将研究T6SS是如何在MDR菌株中被调控的 携带质粒，将我们的结论推广到这些菌株。T6SS的机制 仪器穿过的杀伤者细胞的肽聚糖层还没有被确定 细菌。我们将确定一个假定的肽聚糖酶在这一过程中的作用。我们还有 发现了与质粒丢失相关的表型适应，涉及菌丝和运动性。这个 这些变化的生物学意义将被评估。RNAseq数据引导我们得出了假设 新陈代谢中间体苯乙酸(PAA)被用作化学诱导剂 招募猎物，提高猎物的捕杀效率。确定PAA在T6SS介导中的作用 杀戮可能导致T6SS介导的杀戮具有重要的生态意义的新范式 这意味着什么。这项工作的结果将是对相互作用的详细理解 T6SS和MDR表型以及与此激活相关的生理变化， 这可能会导致开发治疗不动杆菌感染的新策略。

PROJECT SUMMARY/ABSTRACT Multidrug resistant (MDR) Acinetobacter baumannii has emerged as a frequent cause of nosocomial infections with some isolates resistant to all clinically relevant antibiotics. We have previously identified a type VI secretion system (T6SS) in this organism. The multi-component T6SS apparatus facilitates a dynamic contact-dependent injection of toxic effector proteins into competing bacteria. The T6SS is energetically costly, and therefore in most bacteria appears to be exquisitely regulated. We recently showed that several MDR A. baumannii isolates harbor a large, self-transmissible resistance plasmid that negatively regulates T6SS. We found that T6SS is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS- mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. We propose that differentiation of A. baumannii cells into bacterial killers involves multiple phenotypic and metabolic changes and that the fitness costs associated with the MDR and T6SS phenotypes are the driving forces for this differentiation. RNAseq and differential quantitative proteomics experiments revealed that unexpected metabolic pathways related to amino acid catabolism were plasmid-regulated. Most of these metabolic changes seem to be consequence of energetic adaptations to T6SS activation and carriage of a MDR plasmid. By mutagenesis and comparative fitness assays we will determine the importance of these metabolic changes. Interfering with these pathways may result in novel strategies to combat A. baumannii infections. We will investigate how T6SS is regulated in MDR strains that do not carry plasmids to extend our conclusions to these strains. The mechanisms by which the T6SS apparatus crosses the peptidoglycan layer of the killer cell has not been determined in any bacteria. We will define the role of a putative peptidoglycanase in this process. We have also discovered phenotypic adaptations related to plasmid loss, involving piliation and motility. The biological significance of these changes will be assessed. RNAseq data led us to the hypothesis that a metabolic intermediate, phenylacetic acid (PAA), is employed as chemoattractant to recruit prey and increase the killing efficiency. Determining the role of PAA in T6SS mediated killing may result in a new paradigm for T6SS-mediated killing with important ecological implications. The outcome of this work will be a detailed understanding of the interplay between the T6SS and the MDR phenotype and the physiological changes associated to this activation, which may lead to the development of new strategies to treat Acinetobacter infections.