Regulation of Bacteroides fragilis multidrug efflux pumps

描述(由申请人提供)： 前言：尽管关于细菌中抗菌素耐药机制的信息爆炸式增长，但自发现抗生素以来，患者首次死于无法治愈的感染。革兰氏阴性菌多药耐药的主要原因是细菌泵主动外排药物。了解泵是如何调节的是了解MDR发展的关键：细菌只需增加这些外排泵的表达就可以成为MDR。意义：脆弱类杆菌是无处不在的肠道共生菌，当它们逃脱肠道时会导致毁灭性的疾病；它们对抗菌素的耐药性正在显著上升。在需氧菌中，MDR是由转录因子(TF)调节的外排泵引起的。我们假设脆弱杆菌中的MDR外排泵类似地受转录因子的调节。这些转录因子及其调节的泵也可能调节其他毒力因子。退伍军人管理局患者特别有可能受到厌氧菌感染。类杆菌菌血症的死亡率很高(25%-50%)，而肝脏疾病是VA患者中常见的，是死亡率增加的危险因素。我们鉴定了几个MDR临床分离株(包括一株来自阿富汗的GI)，它们的泵活性增加；这些分离株对甲硝唑和其他药物具有耐药性，并分别导致一人死亡和一人截肢。初步研究：在之前的资助期间，我们鉴定并鉴定了脆弱杆菌BME外排泵基因，并表明它们与临床MDR有关(在23篇文献中有描述)；我们发现泵水平和耐药性的增加可以由包括抗菌剂、水杨酸盐、胆汁和群体感应分子在内的各种药剂和应激源诱导。我们构建了新的载体，并对它们进行了修改，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脆弱芽孢杆菌中多药耐药的复杂调控。具体地说，我们现在的目标是：目标1：使用功能基因组学方法确定外排介导的MDR中最重要的转录因子(激活因子和抑制因子)。目的2：确定在外排介导的多药耐药中最重要的转录因子。确定与转铁蛋白相关的表型，测定MIC(最低抑菌浓度)，并确定相关的BME基因。通过物理和功能方法展示约束性。目的：确定外排介导的多药耐药中转铁蛋白调节的临床发生率。检测临床分离株中的转录因子水平，包括最近获得的多药耐药株。确定转移因子对外排介导的多药耐药和生物被膜形成能力的影响。评估胆汁暴露对转铁蛋白转录水平、最低抑菌浓度和生物被膜形成能力的影响。这项研究利用了我们独特的资源：在分子和生化方法方面的专业知识，获得大量临床分离株和相关的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，将揭示有关BF MDR泵调节的重要新信息，并将极大地有助于我们了解这种细菌的调节机制，这种细菌受到与需氧病原体非常不同的压力。脆弱芽孢杆菌是肠道微生物群(一直被称为人体内的一个单独的“器官”)的重要组成部分，被认为具有令人印象深刻的新陈代谢特征，并深刻影响人类健康和疾病的几乎方方面面。在这种全球环境下，从第一批耐多药菌株出现到广泛耐药的时间很短。当无法治愈的感染出现时，争先恐后地开发有效的药物并不是一个可行的公共卫生战略。MDR外流的调节因子是药物发现的重要靶点；识别和了解在这些MDR生物体中最重要的转录因子将导致针对这些因子的表达或翻译的新的有效介入治疗的发展。 公共卫生相关性： 不断上升的抗菌素耐药性仅在美国就造成了50-300亿美元的损失和9万人的生命！多药外排泵在很大程度上造成了这种耐药性。脆弱类杆菌是肠道微生物区系中的重要成员，在人类健康和疾病中起着至关重要的作用。我们几乎没有关于这些泵或它们在这种重要的厌氧病原体中的调节的信息。

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. PUBLIC HEALTH RELEVANCE: Rising antimicrobial resistance costs 5-30 billion dollars and 90,000 lives in the US alone! Multi-drug efflux pumps are largely responsible for this resistance. Bacteroides fragilis is an important member of the gut microbiota that has critical roles in human health and disease. We have virtually no information about these pumps or their regulation in this important anaerobic pathogen.