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

金黄色葡萄球菌涉及80%的所有肌肉骨骼感染（MSKI），费用为17，000 - 150，000美元 每名患者。这些感染中约50%是由耐甲氧西林的S。金黄色葡萄球菌（MRSA） 在医院和社区获得。每次进行的全关节置换（TJR）超过150万例， 年，最严格的预防和无菌手术技术不能降低骨髓炎（OM）率 低于0.5%-2%。治疗已建立的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%。S.金黄色葡萄球菌感染是 这归因于其免疫逃避和抗微生物耐药机制。尽管作出了巨大努力， 尽管开发了解决方案，但治疗模式并没有改善OM患者的不良临床结局， 过去四十年。然而，我们对S.金黄色葡萄球菌定植 小鼠和患者OM过程中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，并提供了一种新的治疗方法， 消除慢性OM的策略。它还回避了有关以下机制的重要问题：1）使 球形S.金黄色葡萄球菌变形为亚微米棒状细菌以侵入OLCN，以及2）使 敏感S. OLCN侵袭后耐抗生素的金黄色葡萄球菌菌株。在过去的四年里，我们 开发了一种新的骨感染芯片，利用硅纳米膜与亚微米（~500 nm）阵列， 模拟OLCN孔（µSiM-CA）。通过靶向缺失候选基因，我们鉴定了细胞壁 青霉素结合蛋白4（pbp 4）是S.金黄色葡萄球菌通过 在体外的µSiM-CA芯片的亚微米通道，然后证明它们抑制OLCN定植， vivo.此外，我们开发并进行了高通量筛选活动，以确定PBP 4抑制剂 （iPBP 4）。在这次更新中，我们将首先证明PBP 4小分子抑制剂（iPBP 4）在 消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 基于基因表达的变化，金黄色葡萄球菌对抗生素的适应性耐受， 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假设。 PBP 4以外的因子，并且OLCN定殖抑制剂的其他化学类别可以通过以下方式鉴定： 经验性地定义遗传决定因素。这些潜在的目标可以用来识别 在单一的筛选方法中使用相应的推定治疗剂。在这个更新计划完成后， CoRTOBI将具有：1）验证最近发现的iPBP 4候选物和潜在的新的独立于PBP的 对OLCN定殖的分子遗传学研究;金黄色葡萄球菌难治性反应 OLCN定殖后的抗生素，以及3）iPBP 4浸渍3D打印的转译方法 骨感染一期翻修手术中的支架。

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.