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

在所有肌肉骨骼感染(MSKI)中，80%与金黄色葡萄球菌有关，费用为17,000-150,000美元 每个病人。这些感染中约有50%是由耐甲氧西林金黄色葡萄球菌(MRSA)引起的 在医院和社区都获得了。总共进行了150万次关节置换(TJR) 一年来，最严格的预防和无菌手术技术并不能降低骨髓炎(OM)的发生率 低于0.5%-2%。治疗已确诊的MSKI仍然具有极大的挑战性，目前的复发率或 翻修手术后持续感染的比例仍高达33%。金黄色葡萄球菌感染的持久性是 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 开发解决方案，治疗范例并没有改善OM患者糟糕的临床结果 过去的四十年里。然而，我们的CoRTOBI范式转换发现金黄色葡萄球菌在 小鼠和患者OM时活体皮质骨的骨细胞陷窝管网络(OLCN)可能是原因之一 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。它还回避了关于以下机制的重要问题：1)使 球形金黄色葡萄球菌变形为亚微米棒状细菌入侵OLCN；2)渲染 金黄色葡萄球菌在OLCN入侵后对抗生素耐药。在过去的四年里，我们 利用亚微米(~500 nm)阵列的硅纳米膜开发了一种新型的骨感染芯片 用于模拟OLCN孔的孔(µSIM-CA)。通过定向删除候选基因，我们确定了细胞壁 转肽酶蛋白，青霉素结合蛋白4(Pbp4)，是金黄色葡萄球菌通过 SIM-CA芯片的亚微米通道，然后证明它们抑制OLCN在体内的定植 活着。此外，我们开发并执行了高通量筛选活动来鉴定pbp4抑制剂。 (IPBP4)。在这次更新中，我们将首先展示PBP4小分子抑制剂(IPBP4)在 在小鼠骨髓炎模型中消除OLCN的侵袭。然后我们将确定OM治疗的靶点 基于使金黄色葡萄球菌对抗生素产生适应性耐受的基因表达变化，在一种新型的µSIM- OLCN芯片平台。最后，我们将测试OLCN殖民可能涉及许多其他 PBP4以外的因素，以及其他化学类别的OLCN定植抑制剂可以通过以下方式确定 经验性地定义遗传决定因素。然后，这些潜在目标可以用来识别 在单一筛查方法中提供相应的推定疗法。在完成这一更新计划时， CoRTOBI将拥有：1)验证最近发现的iPBP4候选对象，以及可能不依赖于PBP的新对象 打击OLCN定植，2)金黄色葡萄球菌耐药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-Print的翻译方法 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.