Regulation of Bacteroides fragilis multidrug efflux pumps

描述（由申请人提供）： 简介：尽管关于细菌抗菌素耐药性机制的信息爆炸，但自抗生素发现以来，患者首次死于无法治愈的感染。革兰阴性菌的多药耐药（MDR）主要是由细菌泵主动外排药物引起的。了解泵是如何调节的是理解MDR发展的关键：细菌可以通过增加这些外排泵的表达而成为MDR。重要性：脆弱拟杆菌是普遍存在的肠道寄生虫，当它们逃离肠道时会引起毁灭性的疾病;它们对抗菌剂的耐药性正在显着上升。在需氧菌中，MDR是由转录因子（TF）调节的外排泵引起的。我们假设MDR外排泵在B。fragilis类似地由TF调节。这些转铁蛋白及其调节的泵也可能调节其他毒力因子。VA患者特别容易发生厌氧菌感染。拟杆菌菌血症的死亡率很高（25- 50%），VA患者常见的肝脏疾病是死亡率增加的危险因素。我们发现了几个MDR临床分离株（包括一个来自阿富汗的GI），其泵活性增加;这些分离株对甲硝唑和其他药物耐药，分别导致1例死亡和1例截肢。初步研究：在前一个资助期，我们确定并表征了B。fragilis bme外排泵基因，并表明它们与临床MDR有关（在23篇出版物中描述）;我们发现，泵水平和耐药性的增加可由多种药物和应激源诱导，包括抗菌剂、水杨酸盐、胆汁和群体感应分子。我们构建了新的载体，并对其进行了修改，以模仿可用于需氧菌的分子工具。研究计划：我们的长期目标是解开B中MDR的复杂调控。脆弱的具体来说，我们现在的目标是：目的1：确定TF最重要的外排介导的MDR（激活和阻遏TF）使用功能基因组学方法。目的2：表征在外排介导的MDR中最重要的TF。确定与TF相关的表型，测量MIC（最小抑制浓度）并确定相关的bme基因。通过物理和功能方法展示约束力。目的3：确定TF调节在外排介导的MDR中的临床流行率。测量临床分离株（包括最近获得的MDR菌株）中的TF转录水平。确定TF对外排介导的MDR和形成生物膜能力的影响。评估胆汁暴露对TF转录水平、MIC和生物膜形成能力的影响。这项研究利用了我们独特的资源：分子和生化方法的专业知识，获得大量临床分离株和相关MIC数据，以及与世界级专家的良好合作关系。一旦完成，这项工作，连同已经进行的研究，将揭示重要的新信息，调节BF MDR泵，并将大大有助于我们的知识，在这种细菌的调节机制，是受到非常不同的压力比好氧病原体。B。脆弱菌是肠道微生物组（其被称为人体内的独立“器官”）的重要组成部分，其被认为具有其自身令人印象深刻的代谢特征，并且深刻地影响人类健康和疾病的几乎每个方面。在这种全球环境下，从第一个MDR菌株出现到广泛耐药几乎没有时间。当出现无法治愈的感染时，竞相开发有效的药物不是一种可行的公共卫生战略。MDR外排的调节因子是药物发现的重要靶点;识别和理解这些MDR生物体中最重要的TF将导致开发针对这些因子表达或翻译的新的有效的介入疗法。

DESCRIPTION (provided by applicant): Introduction: Despite an information explosion about antimicrobial resistance mechanisms in bacteria, patients are dying of untreatable infections for the first time since antibiotics were discovered. The main cause of multidrug resistance (MDR) in gram-negative bacteria is active efflux of drug by bacterial pumps. Knowing how the pumps are regulated is the key to understanding MDR development: bacteria can become MDR simply by increasing expression of these efflux pumps. Significance: Bacteroides fragilis are ubiquitous gut commensals that cause devastating disease when they escape the gut; their resistance to antimicrobials is rising significantly. In aerobes, MDR is caused by efflux pumps regulated by transcription factors (TFs). We hypothesize that MDR efflux pumps in B. fragilis are similarly regulated by TFs. These TFs, and the pumps they regulate, may also regulate other virulence factors. VA patients are particularly at risk for anaerobic infections. Mortality in Bacteroides bacteremia is high (25- 50%) and liver disease, common in VA patients, is a risk factor for increased mortality. We identified several MDR clinical isolates (including one from a GI in Afghanistan) with increased pump activity; these isolates were resistant to metronidazole and other agents and resulted in one death and one amputation, respectively. Preliminary Studies: In the previous funding period, we identified and characterized the B. fragilis bme efflux pump genes and showed that they were implicated in clinical MDR (described in 23 publications); we found that increases in pump levels and in resistance could be induced by a variety of agents and stressors including antimicrobials, salicylate, bile and quorum sensing molecules. We constructed new vectors and modified them to mimic the molecular tools available for use with aerobes. Research Plan: Our long term goal is to unravel the complex regulation of MDR in B. fragilis. Specifically, our goals now are to: AIM 1: Identify the TFs most important in efflux-mediated MDR (both activator and repressor TFs) using a functional genomic approach. AIM 2: Characterize TFs most important in efflux-mediated MDR. Determine the phenotype associated with the TF, measure MICs (minimal inhibitory concentrations) and determine the relevant bme genes. Demonstrate binding by physical and functional approaches. AIM 3: Determine the clinical prevalence of TF regulation in efflux mediated MDR. Measure TF transcription levels in clinical isolates, including the recently acquired MDR strains. Determine the effect of TF on efflux-mediated MDR and on ability to form biofilms. Assess the effect of bile exposure on TF transcription levels, MICs and biofilm forming ability. This study takes advantage of our unique resources: expertise with molecular and biochemical approaches, access to a massive collection of clinical isolates and associated MIC data, and excellent collaborative relationships with world-class experts. Once completed, this work, together with the studies already conducted, will reveal important new information about regulation of BF MDR pumps and will greatly contribute to our knowledge of regulation mechanisms in this bacterium that is subject to very different stresses than aerobic pathogens. B. fragilis is an important component of the gut microbiome (which has been called a separate "organ" within the human body) that is recognized as having its own impressive metabolic profile and profoundly affects almost every aspect of human health and disease. In this global environment, there is little time from the emergence of the first MDR strains to widespread resistance. Racing to develop effective drugs when untreatable infections emerge is not a viable public health strategy. Regulators of MDR efflux are important targets in drug discovery; identifying and understanding the TFs that are most important in these MDR organisms will lead to the development of new and effective interventional therapies that target the expression or translation of these factors.