Elucidating the Mechanisms of S. aureus Motility in Bone and Developing Interventions

阐明金黄色葡萄球菌在骨中的运动机制并制定干预措施

• 批准号: 10402966
• 负责人: Hani A Awad
• 金额: $ 35.97万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-20 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10402966
• 关键词: 3D Print; Adhesions; Adjuvant; Antibiotic Therapy; Antibiotics; Antimicrobial Resistance; Bacteria; Bacterial Adhesins; Biological Process; Candidate Disease Gene; Cell Wall; Cell division; Chemicals; Chronic; Clinical; Communities; Cues; Disease; Dose; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic Determinism; Genomics; Hospitals; Immune Evasion; Implant; In Vitro; Infection; Intervention; Invaded; Libraries; Local Therapy; Membrane; Methods; Microfluidics; Minimum Inhibitory Concentration measurement; Modeling; Molecular; Molecular Genetics; Mus; Musculoskeletal; Operative Surgical Procedures; Osteocytes; Osteomyelitis; Outcome; Pathway interactions; Patients; Penicillin Binding Protein 4; Peptidoglycan; Peptidyltransferase; Polymethyl Methacrylate; Process; Prophylactic treatment; Proteins; Refractory; Replacement Arthroplasty; Rod; Role; Silicon; Specificity; Staphylococcus aureus; Staphylococcus aureus infection; Surface; Systemic Therapy; Techniques; Testing; Therapeutic; Translational Research; Translations; Vancomycin; Work; antibiotic tolerance; base; bone; bone healing; cell motility; cellular targeting; chronic infection; cortical bone; cost; design; efficacy validation; high throughput screening; in vivo; inhibitor; methicillin resistant Staphylococcus aureus; microbial; microchip; mouse model; mutant; nanomembrane; novel; novel therapeutic intervention; osteoimmunology; programs; recurrent infection; resistance mechanism; response; scaffold; screening; small molecule; small molecule inhibitor; submicron; therapeutic candidate; transcriptome sequencing; translational impact

Staphylococcus aureus is involved in 80% of all musculoskeletal infections (MSKI) costing $17,000–$150,000 per patient. Approximately 50% of these infections are caused by methicillin-resistant S. aureus (MRSA) acquired in both hospital and community. With >1.5 million total joint replacements (TJR) performed each year, the most rigorous prophylaxis and aseptic surgical techniques cannot reduce osteomyelitis (OM) rates below 0.5%–2%. Treating established MSKI remains extremely challenging, with current rates of recurrent or persistent infection following revision surgery still as high as 33%. The persistence of S. aureus infection is attributed to its arsenal of immune evasion and antimicrobial resistance mechanisms. Despite great efforts to develop solutions, treatment paradigms have not improved the poor clinical outcomes for OM patients over the last four decades. However, our CoRTOBI paradigm-shifting discovery of S. aureus colonization of the osteocyte lacuno-canalicular network (OLCN) of live cortical bone during OM in mice and patients may explain why previous approaches for treating recurring bone infections have failed, and provide a new therapeutic strategy for eliminating chronic OM. It also begs important questions about the mechanisms that: 1) enable spherical S. aureus to deform into submicron-rod shaped bacteria to invade the OLCN, and 2) render susceptible S. aureus strains refractory to antibiotics after OLCN invasion. Over the past four years we developed a novel bone infection-on-chip utilizing silicon nanomembrane with submicron (~500 nm) array of pores to simulate OLCN orifices (µSiM-CA). By targeted deletion of candidate genes, we identified cell wall transpeptidase proteins, penicillin binding protein 4 (Pbp4), as essential for S. aureus propagation through submicron channels of the µSiM-CA chips in vitro and then demonstrated that they inhibit OLCN colonization in vivo. Moreover, we developed and performed a high throughput screening campaign to identify PBP4 inhibitors (iPBP4). In this renewal, we will first demonstrate the efficacy of PBP4 small molecule inhibitors (iPBP4) in abrogating the OLCN invasion in mouse models of osteomyelitis. We will then identify targets for OM therapy based on gene expression changes that affords S. aureus adaptive tolerance to antibiotics in a novel µSiM- OLCN Chip platform. Finally, we will test the premise that OLCN colonization likely involves many additional factors other than PBP4, and that other chemical classes of OLCN colonization inhibitors can be identified by empirically defining the genetic determinants. These potential targets can then be used to identify corresponding putative therapeutics in a single screening approach. At the completion of this renewal program, CoRTOBI will have: 1) validated recently discovered iPBP4 candidates and potentially new PBP-independent hits against OLCN colonization, 2) a molecular genetic understanding of S. aureus refractory response to antibiotics following OLCN colonization, and 3) translational methods for iPBP4 impregnated 3D-printed scaffolds in one-stage revision surgery for bone infections.

80% 的肌肉骨骼感染 (MSKI) 涉及金黄色葡萄球菌，造成的损失为 17,000 美元至 150,000 美元 每个病人。大约 50% 的感染是由耐甲氧西林金黄色葡萄球菌 (MRSA) 引起的 医院和社区均获得。每次进行的总关节置换术 (TJR) 均超过 150 万次 年，最严格的预防和无菌手术技术无法降低骨髓炎 (OM) 率 低于 0.5%–2%。治疗已形成的 MSKI 仍然极具挑战性，目前的复发率或 翻修手术后持续感染仍高达33%。金黄色葡萄球菌感染的持续性是 归因于其免疫逃避和抗菌素耐药机制。尽管付出了巨大的努力 制定解决方案，治疗模式并没有改善 OM 患者的不良临床结果 过去四十年。然而，我们的 CoRTOBI 范式转变发现金黄色葡萄球菌定植于 小鼠和患者 OM 期间活皮质骨的骨细胞腔隙小管网络 (OLCN) 可以解释 为什么以前治疗复发性骨感染的方法失败了，并提供了新的治疗方法 消除慢性 OM 的策略。它还引出了有关以下机制的重要问题：1） 球形金黄色葡萄球菌变形为亚微米杆状细菌侵入 OLCN，2) 渲染 OLCN 入侵后对抗生素耐药的敏感金黄色葡萄球菌菌株。过去四年我们 开发了一种新型骨感染芯片，利用具有亚微米（~500 nm）阵列的硅纳米膜 模拟 OLCN 孔口 (μSiM-CA) 的孔。通过靶向删除候选基因，我们鉴定了细胞壁 转肽酶蛋白、青霉素结合蛋白 4 (Pbp4)，对于金黄色葡萄球菌通过 µSiM-CA 芯片的亚微米通道在体外，然后证明它们抑制 OLCN 定植 体内。此外，我们开发并进行了高通量筛选活动来鉴定 PBP4 抑制剂 （iPBP4）。在本次更新中，我们将首先证明 PBP4 小分子抑制剂（iPBP4）在 消除小鼠骨髓炎模型中的 OLCN 侵袭。然后我们将确定 OM 治疗的目标 基于基因表达变化，使金黄色葡萄球菌在新型 µSiM- 中对抗生素具有适应性耐受性 OLCN芯片平台。最后，我们将测试 OLCN 殖民化可能涉及许多额外的前提 PBP4 以外的因素，以及 OLCN 定植抑制剂的其他化学类别可以通过以下方式鉴定： 凭经验定义遗传决定因素。然后可以使用这些潜在目标来识别 在单一筛选方法中相应的假定疗法。在本次更新计划完成后， CoRTOBI 将具有：1) 经过验证的最近发现的 iPBP4 候选者和潜在的新的独立于 PBP 的 对抗 OLCN 定植，2) 对金黄色葡萄球菌难治反应的分子遗传学理解 OLCN 定植后的抗生素，以及 3) iPBP4 浸渍 3D 打印的转化方法 用于骨感染一期翻修手术的支架。