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.

金黄色葡萄球菌涉及所有肌肉骨骼感染（MSKI）的80％，费用为$ 17,000- $ 150,000 每个患者。这些感染中约有50％是由金黄色葡萄球菌（MRSA）引起的 在医院和社区中获得。 > 150万的总置换量（TJR）执行了 一年，最严格的预防和无菌外科手术技术无法降低骨髓炎（OM）率 低于0.5％–2％。当前的经常性率或 修订手术后的持续感染仍然高达33％。金黄色葡萄球菌感染的持久性是 归因于其免疫进化和抗菌耐药机制的武器库。尽管做出了巨大的努力 开发解决方案，治疗范例并没有改善OM患者的临床结果不佳 最近四十年。但是，我们的Cortobi范式转移发现了金黄色葡萄球菌殖民 OM在小鼠中，活皮质骨的骨细胞lacuno-canalicular网络（OLCN），患者可能会解释 为什么以前治疗重复骨感染的方法失败了，并提供了一种新的疗法 消除慢性OM的策略。它还提出了有关以下机制的重要问题：1）启用 球形金黄色葡萄球菌变形为亚微米杆形细菌以侵入OLCN，2）渲染 OLCN入侵后，敏感的金黄色葡萄球菌菌株对抗生素难治性。在过去的四年中 开发了一种新型的片骨感染，利用硅纳米膜和亚微米（〜500 nm）阵列 孔以模拟OLCN孔（µSIM-CA）。通过针对候选基因的靶向缺失，我们确定了细胞壁 转肽酶蛋白，青霉素结合蛋白4（PBP4），对于金黄色葡萄球菌传播至关重要 µSIM-CA芯片的亚微米通道在体外，然后证明它们抑制了OLCN定植 体内。此外，我们开发并进行了高吞吐量筛选活动，以识别PBP4抑制剂 （IPBP4）。在此续签中，我们将首先证明PBP4小分子抑制剂（IPBP4）在 废除骨髓炎小鼠模型中的OLCN侵袭。然后，我们将确定OM治疗的目标 基于基因表达变化，在新型µSIM-中具有金黄色葡萄球菌自适应耐受性 OLCN芯片平台。最后，我们将测试OLCN殖民化可能涉及许多其他的前提 PBP4以外的其他因素，其他化学类别的OLCN定殖抑制剂可以通过 从经验上定义遗传决定剂。然后可以使用这些潜在目标来识别 在单个筛选方法中相应的推定理论。在完成此续签计划时， Cortobi将有：1）最近发现的IPBP4候选物以及潜在的不依赖PBP的验证 对OLCN定植的命中，2）对金黄色葡萄球菌难治反应的分子遗传理解 OLCN定殖后的抗生素和3）IPBP4的翻译方法浸渍了3D打印 骨骼感染的一阶段翻修手术中的脚手架。