Targeting cytochrome bd as an anti-biofilm strategy

靶向细胞色素 bd 作为抗生物膜策略

• 批准号: 10642243
• 负责人: Maria Hadjifrangiskou
• 金额: $ 27.56万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-08 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10642243
• 关键词: Address; Aerobic; Affect; Affinity; Anaerobic Bacteria; Antibiotic Therapy; Antibiotics; Architecture; Attenuated; Bacteria; Bacterial Infections; Biology; Bladder; Catheterization; Catheters; Cell Respiration; Cells; Cellulose; Chemicals; Chemosensitization; Code; Collaborations; Complex; Cytochromes; Cytosol; Defect; Effectiveness; Environment; Epithelial Cells; Epithelium; Escherichia coli; Exhibits; Extracellular Matrix; Genes; Goals; Growth; Heme; Hypoxia; Impairment; In Vitro; Individual; Infection; Infective cystitis; Innate Immune Response; Invaded; Link; Membrane; Membrane Potentials; Membrane Proteins; Microbial Biofilms; Minimum Inhibitory Concentration measurement; Mitochondria; Modeling; Molecular; Mus; Oxidases; Oxygen; Pathogenesis; Predisposition; Prevention strategy; Process; Proteins; Proton Pump; Proton-Motive Force; Pump; Respiration; Role; Surface; Testing; Therapeutic; Urinary tract; Urinary tract infection; Urologic Diseases; Uropathogenic E. coli; Virulence Factors; Work; bacterial community; cell community; cell motility; copper oxidase; cytochrome c; cytochrome c oxidase; efficacy testing; inhibitor; insight; mouse model; mutant; novel therapeutics; overexpression; pathogen; preclinical evaluation; prevent; quinol oxidase; respiratory; stress tolerance; therapeutic development; three dimensional structure; urinary

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.

总结 意义：尿路感染（UTI）是最常见的泌尿系统疾病之一， 主要由尿路致病性大肠杆菌（UPEC）引起。UPEC引起的膀胱感染的特点是短暂的 细胞内阶段，在此期间细菌侵入表面上皮（小面）细胞并在胞质溶胶内分裂， 形成称为生物膜的多细胞群落。在细胞质中复制后，细菌离开细胞内生物膜 - 在此过程中杀死膀胱上皮细胞-并传播到幼稚的小面细胞或上尿路 道。当处于细胞内生物膜状态时，细菌逃避先天免疫应答和免疫抑制剂的作用。 抗生素类似地，在导管插入的个体中，导管表面上的生物膜的形成产生额外的生物降解。 UPEC的保护性生态位，它可以传播到膀胱和种子感染。这个目标 建议是评估通过干扰UPEC呼吸来抑制生物膜的潜力。 原理和假设：虽然UPEC是兼性厌氧菌，但它们在感染过程中呼吸氧气， 缺氧的膀胱环境。有氧呼吸和氧感也与 关键UPEC毒力因子的表达。我们以前已经表明，有氧呼吸是必不可少的， UPEC建立感染。在由UPEC编码的三种呼吸性醌醇氧化酶中，细胞色素bd具有 对分子氧的亲和力最高，超过线粒体细胞色素c的亲和力1000倍。删除 编码细胞色素bd的cydABX基因中的一个，在体外不赋予生长缺陷，但导致显著的 UPEC生物膜结构的改变，导致生物膜状态下对抗生素的敏感性更高。 此外，cydABX缺失突变体是不运动的，表现出降低的质子动力（pmf），并且是不稳定的。 在鼠UTI模型中减毒。最后，cydABX的缺失导致低亲和力的表达增加。 醌醇氧化酶细胞色素b 0。我们假设细胞色素bd可以化学靶向阻止 生物膜形成或从预形成的生物膜传播。我们进一步证明细胞色素bd具有 一种促进运动的作用。我们提出了两个目标来检验提出的假设： 目的：目的1将评价使用已知细胞色素bd抑制剂靶向细胞色素bd是否可以 提高抗生素的有效性。目的2将确定是否降低膜电位的 细胞色素bd突变体对pmf依赖性过程产生普遍或特异性影响，并确定如何 这些受损的过程影响运动性。最后，使用细胞色素bd的化学抑制，我们将评估 细胞色素BD功能的丧失是否损害了从生物膜的传播。 影响：这些研究将是第一个解决细胞色素bd对UPEC运动的独特贡献， 将确定是否针对细菌呼吸是一个可行的治疗或预防策略对尿路感染。