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.

概括 意义：尿路感染（UTI）是最普遍的泌尿科疾病之一，它是引起的 主要是由尿道病大肠杆菌（UPEC）。 UPEC感染膀胱感染的特征是瞬态 细胞内阶段细菌侵入浅表上皮（刻面）细胞并在细胞质内分裂为 形成称为生物膜的多细胞社区。在细胞质中复制后，细菌退出细胞内生物膜 - 在此过程中杀死膀胱上皮细胞 - 并传播到幼稚的小片细胞或上尿 道。在细胞内生物膜状态下，细菌逃避了先天免疫反应和 抗生素。同样，在导管个体中，导管表面生物膜的形成也会产生额外的 UPEC的保护性利基市场可以从中传播到膀胱和种子感染。目标的目标 建议是通过干扰UPEC呼吸来评估抑制生物膜的潜力。 理由和假设：尽管UPEC是兼性厌氧菌，但它们在感染过程中呼吸氧气 低氧膀胱环境。有氧呼吸和氧气感应也已与 临界UPEC病毒因子的表达。我们以前已经表明，有氧呼吸对于 UPEC建立感染。在UPEC编码的三种呼吸道喹酚氧化物中，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的亲和力高1000倍。删除 在为细胞色素BD编码的CYDABX基因中，在体外不会赋予生长缺陷，而是导致显着的 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缺失突变体是非运动的，暴露于质子运动力（PMF），为 在鼠类UTI模型中衰减。最后，cydabx的缺失导致低亲和力的表达增加 奎诺氧化物细胞色素B0。我们假设细胞色素BD可以化学靶向阻碍 从预先形成的生物膜中形成生物膜或传播。我们进一步确认细胞色素BD有 在激动能力中的作用。我们提出了两个目的，以检验定位的假设： 目的：将评估AIM 1是否使用已知的细胞色素BD抑制剂靶向细胞色素BD是否可以 提高抗生素有效性。 AIM 2将确定膜的潜力降低 细胞色素BD突变体对PMF依赖性过程赋予广义或特定影响，并确定如何 这些受损的过程会影响运动。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传播。 影响：这些研究将是第一个解决细胞色素BD对UPEC运动和 将确定靶向细菌呼吸是针对UTI的可行治疗或预防策略。