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

氯酸盐治疗铜绿假单胞菌慢性伤口感染的生物学机制和后果

• 批准号: 9810001
• 负责人: Dianne K Newman
• 金额: $ 21.46万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810001
• 关键词: Active Biological Transport; Address; Affect; Aminoglycosides; Amputation; Anaerobic Bacteria; Antibiotics; Bacteria; Biochemical; Biological; Burn injury; CCRL2 gene; Cell Death; Cell division; Cell membrane; Cells; Charge; Chlorates; Chronic; Collaborations; Cystic Fibrosis; Data; Development; Diabetic Foot Ulcer; Diabetic mouse; Diabetic ulcer; Dimensions; Drug Targeting; Effectiveness; Enzymes; Exploratory/Developmental Grant; Exposure to; Eye Infections; Foundations; Gene Expression; Genes; Genetic; Genetic Screening; Hodgkin Disease; Homologous Gene; Hypoxia; In Situ; In Vitro; Infection; Knowledge; Laboratories; Link; Lower Extremity; Lung; Malignant neoplasm of prostate; Mammals; Measures; Mediating; Metabolism; Microbe; Microbial Biofilms; Modeling; Molecular; Nitrate Reductases; Nitrates; Outcome; Pathway interactions; Patients; Pharmaceutical Preparations; Physiological; Population; Prodrugs; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Publishing; Research; Resistance; Respiration; Techniques; Testing; Time; Tobramycin; Topical application; Toxic effect; Work; Wound Healing; Wound Infection; acute infection; analog; antibiotic tolerance; base; chronic infection; chronic wound; experimental study; follow-up; foot; healing; in vivo; inhibitor/antagonist; insight; malignant breast neoplasm; mortality; mouse model; mutant; non-healing wounds; novel; pathogen; pathogenic bacteria; polymicrobial biofilm; protein aggregation; protein folding; transposon sequencing; ventilator-associated pneumonia

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.

项目总结 铜绿假单胞菌是一种在急性感染(烧伤、伤口、呼吸机)中发现的条件致病菌 相关肺炎、眼部感染)和慢性足部感染(糖尿病溃疡)和肺部慢性感染(囊性 纤维化)(1)。这种细菌通常以生物膜的形式存活在这些环境中，形成和高水平 对抗生素的耐受性干扰了有效的患者治疗。低氧/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 目前存在针对这一顽固群体的目标。慢性无法愈合的伤口通常含有生物膜 包括兼性厌氧菌，如铜绿假单胞菌，每年影响全球数百万人(4)。 仅糖尿病足溃疡就占非创伤性下肢截肢的80%，并与 5年死亡率为43-55%，高于霍奇金氏病、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同源基因(7)。我们最近的体外研究表明，氯酸盐可以特异性地杀死缺氧/缺氧 通过利用NAR活性来产生有毒产品的生物膜亚群(8)。这样做的总体目标是 建议更好地了解氯酸盐毒性的机制，并评估其作为一种促进药物在 慢性创面糖尿病小鼠模型。我们的假设是氯酸盐杀死缺氧生物膜亚群 因为它触发了蛋白质聚集，体内生物膜的发展依赖于NAR的表达，而且， 因此，局部使用氯酸盐将促进伤口愈合。我们的假设已经公式化了 根据初步数据显示，接触氯酸盐上调了涉及蛋白质折叠和蛋白质合成的基因 铜绿假单胞菌在小鼠模型中发育成生物膜聚集体，其尺寸将是 预计包括表达NAR的低氧/缺氧亚群。在目标1中，我们将使用目标 用生物化学方法检验氯酸盐毒性与蛋白质聚集有关的假说，以及一种非生物化学方法。 偏向遗传TN-SEQ筛选以确定硝酸盐充足条件下氯酸盐抗性的决定因素。在……里面 目的2，我们将评估铜绿假单胞菌在体内是否表达NAR，以及它的表达是否需要 生物膜的发育，并可被氯酸盐劫持，以杀死耐药生物膜亚群，从而 加速伤口愈合。建议的研究具有重要意义，因为它将提供新的基础 了解氯酸盐毒性的基础机制，并使我们能够更好地评估氯酸盐 在感染的情况下，有可能成为针对铜绿假单胞菌生物被膜的有效前药。长期的 这项研究产生的结果可能会为开发氯酸盐提供可能相关的见解 作为一种治疗不同感染中的多菌生物膜的新方法。