Targeting cytochrome bd as an anti-biofilm strategy

摘要 意义：尿路感染(UTI)是最常见的泌尿系统疾病之一，由 主要由尿路致病性大肠埃希菌(UPEC)引起。UPEC膀胱感染的特征是一过性 细菌侵入表面上皮(小平面)细胞并在胞质内分裂以 形成称为生物膜的多细胞群落。细菌在胞浆中复制后，离开细胞内的生物膜。 -在此过程中杀死膀胱上皮细胞-并扩散到幼稚的小关节细胞或上尿路 一条小路。而在细胞内生物膜状态下，细菌逃避先天免疫反应和 抗生素。同样，在留置尿管的个体中，导尿管表面生物膜的形成产生了额外的 UPEC的保护性生态位，从那里它可以传播到膀胱和种子感染。这样做的目的是 建议评估通过干扰UPEC呼吸来抑制生物被膜的潜力。 理论基础和假设：虽然UPEC是兼性厌氧菌，但它们在感染过程中呼吸氧气 低氧的膀胱环境。有氧呼吸和氧气感应也与 UPEC毒力关键因子的表达。我们之前已经证明，有氧呼吸对于 UPEC以确定感染。在UPEC编码的三种呼吸性喹酚氧化酶中，细胞色素BD具有 对分子氧的亲和力最高，比线粒体细胞色素c的亲和力高1000倍。删除 在编码细胞色素BD的cydABX基因中，在体外不会导致生长缺陷，但会导致显著的 UPEC生物膜结构的改变，导致生物膜状态下对抗生素的更高敏感性。 此外，cydABX缺失突变体是非运动性的，表现出质子动力(PMF)降低和 在小鼠尿路感染模型中衰减。最后，cydABX的缺失导致低亲和力的表达增加 苯二酚氧化酶细胞色素b0。我们假设细胞色素BD可以被化学靶向阻挠 从预先形成的生物膜形成或扩散的生物膜。我们进一步假设细胞色素BD具有 在激励动力方面的作用。我们提出了两个目的来检验所提出的假设： 目的：AIM 1将评估使用已知的细胞色素BD抑制剂靶向细胞色素BD是否可以 提高抗生素的有效性。目标2将确定细胞膜电位的降低是否 细胞色素BD突变体在PMF依赖的过程中给予全身性或特异性影响并确定如何 这些受损的过程会影响运动能力。最后，使用化学抑制细胞色素BD，我们将评估 细胞色素BD功能的丧失是否会损害生物膜的扩散。 影响：这些研究将首次解决细胞色素BD对UPEC运动和 将决定以细菌呼吸为靶点是治疗或预防尿路感染的可行策略。

SUMMARY Significance: Urinary tract infection (UTI) is among the most prevalent urologic diseases, and it is caused primarily by uropathogenic Escherichia coli (UPEC). Bladder infection by UPEC is characterized by a transient intracellular stage during which bacteria invade superficial epithelial (facet) cells and divide within the cytosol to form multicellular communities called biofilms. After replicating in the cytosol, bacteria exit the intracellular biofilm – killing the bladder epithelial cell in the process – and disseminate to naïve facet cells or to the upper urinary tract. While in the intracellular biofilm state, bacteria evade innate immune responses and the effects of antibiotics. Similarly, in catheterized individuals, formation of biofilm on the catheter surface creates an additional protective niche for UPEC, from which it can disseminate to the bladder and seed infection. The goal of this proposal is to evaluate the potential of inhibiting biofilm by interfering with UPEC respiration. Rationale and Hypothesis: Although UPEC are facultative anaerobes, they respire oxygen during infection in the hypoxic bladder environment. Aerobic respiration and oxygen sensing have also been linked to the expression of critical UPEC virulence factors. We have previously shown that aerobic respiration is essential for UPEC to establish infection. Of the three respiratory quinol oxidases encoded by UPEC, cytochrome bd has the highest affinity for molecular oxygen, exceeding the affinity of mitochondrial cytochrome c by 1000-fold. Deletion of the cydABX genes that code for cytochrome bd, does not impart a growth defect in vitro, but leads to significant alterations in UPEC biofilm architecture, leading to higher susceptibility to antibiotics in the biofilm state. Furthermore, cydABX deletion mutants are non-motile, exhibit decreased proton motive force (pmf) and are attenuated in a murine UTI model. Finally, deletion of cydABX results in increased expression of the low affinity quinol oxidase cytochrome b0. We hypothesize that cytochrome bd can be chemically targeted to thwart biofilm formation or dissemination from a pre-formed biofilm. We further posit that cytochrome bd has a role in energizing motility. We propose two aims to test the posed hypotheses: Aims: Aim 1 will will evaluate whether targeting of cytochrome bd using known cytochrome bd inhibitors can enhance antibiotic effectiveness. Aim 2 will determine whether the decreased membrane potential of the cytochrome bd mutant imparts generalized or specific effects on pmf-dependent processes and determine how these impaired processes affect motility. Finally, using chemical inhibition of cytochrome bd we will evaluate whether loss of cytochrome bd function impairs dissemination from the biofilm. Impact: These studies will be the first to address the unique contribution of cytochrome bd on UPEC motility and will determine whether targeting bacterial respiration is a viable therapeutic or prevention strategy against UTI.