Biological mechanisms and consequences of chlorate treatment on Pseudomonas aeruginosa chronic wound infections

项目摘要 铜绿假单胞菌是一种机会致病菌，在急性感染（烧伤，创伤，呼吸机 相关性肺炎、眼部感染）以及足部（糖尿病溃疡）和肺部（囊性 纤维化）（1）。这种细菌通常以生物膜的形式在这些环境中生存，其形成和高水平的 其抗生素耐受性干扰有效的患者治疗。缺氧/缺氧，生长缓慢 生物膜核心定义了对常规药物最耐受的亚群（2，3）。然而，很少有治疗方法 目前存在的目标是这个非法组织。慢性不愈合伤口通常含有生物膜 包括兼性厌氧菌如铜绿假单胞菌，并且每年影响全世界数百万人（4）。 仅糖尿病足溃疡就占非创伤性下肢截肢的80%，并且与糖尿病足溃疡相关。 5年死亡率为43- 55%，高于霍奇金病、乳腺癌或前列腺癌（5）。 nar介导的硝酸盐呼吸是细菌病原体广泛使用的厌氧代谢（6）; 哺乳动物缺乏Nar同系物（7）。我们最近的体外研究表明，氯酸盐特异性地杀死缺氧/缺氧 生物膜亚群通过选择Nar活性产生毒性产物（8）。本报告的总体目标 建议是为了更好地了解氯酸盐毒性的机制，并评估其作为前药的效用， 慢性创伤糖尿病小鼠模型。我们的假设是氯酸盐杀死缺氧生物膜亚群 因为它触发蛋白质聚集，生物膜在体内的发展取决于Nar表达，并且， 因此，氯酸盐的局部应用将促进伤口愈合。我们的假设是 初步数据显示，氯酸盐暴露上调了蛋白质折叠相关基因， 铜绿假单胞菌在小鼠模型中发展成生物膜聚集体，其尺寸 预期包含表达Nar的缺氧/缺氧亚群。在目标1中，我们将使用目标 生物化学方法来测试假设，氯酸盐毒性与蛋白质聚集，和一个联合国， 偏向遗传Tn-seq筛选，以鉴定在硝酸盐充足条件下氯酸盐抗性的决定因素。在 目的2，我们将评估铜绿假单胞菌是否在体内表达Nar，以及它的表达是否是 生物膜的发展，并可以劫持氯酸盐杀死抗生素耐受性生物膜亚群，因此 加速伤口愈合这项研究具有重要意义，因为它将提供新的基础。 了解氯酸盐毒性的机制，使我们能够更好地评估氯酸盐的毒性。 潜在的是一种有效的前体药物，在感染的情况下，目标铜绿假单胞菌生物膜。长期 这项研究产生的结果可能会提供与开发氯酸盐相关的见解 作为不同感染中多微生物生物膜的新型治疗。

PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen found in acute infections (burns, wounds, ventilator associated pneumonia, eye infections) and chronic infections of the foot (diabetic ulcers) and lung (cystic fibrosis) (1). This bacterium commonly survives in these contexts as a biofilm, the formation and high-level antibiotic tolerance of which interferes with effective patient treatment. The hypoxic/anoxic, slowly-growing biofilm core defines the subpopulation that is most tolerant of conventional drugs (2, 3). Yet few therapies currently exist that target this recalcitrant group. Chronic nonhealing wounds typically contain biofilms comprising facultative anaerobes such as P. aeruginosa, and affect millions of people annually worldwide (4). Diabetic foot ulcers alone contribute to 80% of nontraumatic lower-extremity amputations and are associated with 5-year mortality rates of 43-55%, higher than Hodgkin's disease, breast cancer or prostate cancer (5). Nar-mediated nitrate respiration is a widespread anaerobic metabolism used by bacterial pathogens (6); mammals lack a Nar homolog (7). Our recent in vitro work shows that chlorate specifically kills hypoxic/anoxic biofilm subpopulations by co-opting Nar activity to produce a toxic product (8). The overall objective of this proposal is to better understand the mechanism of chlorate toxicity and assess its utility as a pro-drug in a chronic wound diabetic mouse model. Our hypothesis is that chlorate kills anoxic biofilm subpopulations because it triggers protein aggregation, that biofilm development in vivo depends on Nar expression, and, therefore, that topical application of chlorate will facilitate wound healing. Our hypothesis has been formulated based on preliminary data that showed chlorate exposure upregulates genes involved in protein folding and that P. aeruginosa develops into biofilm aggregates in the mouse model with dimensions that would be expected to comprise hypoxic/anoxic subpopulations expressing Nar. In Aim 1, we will use targeted biochemical approaches to test the hypothesis that chlorate toxicity is linked to protein aggregation, and an un- biased genetic Tn-seq screen to identify determinants of chlorate resistance under nitrate-replete conditions. In Aim 2, we will assess whether P. aeruginosa expresses Nar in vivo, and whether its expression is required for biofilm development and can be hijacked by chlorate to kill antibiotic tolerant biofilm subpopulations, thus accelerating wound healing. The proposed research is significant because it will provide new basic understanding of the mechanisms underpinning chlorate toxicity, and allow us to better evaluate chlorate's potential to be an effective pro-drug to target P. aeruginosa biofilms in the context of infection. The long-term outcomes generated by this research are likely to provide insights that may be relevant to developing chlorate as a novel treatment for polymicrobial biofilms in disparate infections.