Project 1: Elucidating the Mechanisms of S. aureus Motility in Bone and Developing Interventions

摘要 假体关节感染(PJI)，其中绝大多数是由葡萄球菌引起的，是 择期全关节置换术。独有问题的致病机制 导致这些感染无法治愈的PJI在很大程度上仍不为人所知。为了解决这一知识差距，我们 进行了广泛的透射电子显微镜(TEM)研究，发现了以前 未报道的金黄色葡萄球菌在皮质骨小管和亚微米裂缝中定植的机制。我们的 与广为接受的教条相反，新奇的观察表明，金黄色葡萄球菌肯定有能动性。 使其能够通过3D的几何和刚性线索识别小管和亚微米裂缝的机制 骨的细胞外基质，随后从球形球菌变形为杆状细菌，推动 它的有丝分裂后代通过亚微米小管的不对称隔面。这一机制 将金黄色葡萄球菌保护在这些亚微米级的裂缝和小管中，使白细胞无法 联系到他们。这也可能限制了抗菌剂的有效性，并使感染无法治愈。 因此，我们的全球假设是金黄色葡萄球菌利用趋触性和趋多性(定向迁移率由 分别来自3D细胞外基质的几何和刚性线索)，以不可治愈地定植小管和 皮质骨中的亚微米通道。在这个应用中，我们采用了创新的基因和小分子 设计抑制触觉趋化和多趋性的新一代抗菌剂的筛选方法 皮质骨的介导性定植。在目标1中，我们使用了创新的纳米多孔硅膜传输井 确定金黄色葡萄球菌在体外通过亚微米通道的占据和迁移的动力学 通过小管模拟体内的趋上性和趋多性。我们还建议完成一项病例对照 临床相关研究记录金黄色葡萄球菌在微裂缝和骨细胞-管状网络中的定植 受感染的人类皮质骨。在目标2中，我们采取有重点的候选基因分析和无偏见的从头开始 基因筛选与互补经验TnSeq突变文库筛选方法鉴定金黄色葡萄球菌 参与小管侵袭和迁移的基因。在目标3中，我们建议开发3D打印间隔器 注入了新型抗生素，目标是生物膜中合成RNA和蛋白质的关键酶- 相关细菌或参与趋上性和趋多性的基本蛋白质，以证明其疗效 在已建立的OM小鼠模型中对脓毒症股骨钢板进行单期翻修。多学科的 方法，包括组织工程和3D打印、微生物基因组学和高通量 筛选小分子抗菌剂，将提供解决重大问题所需的关键信息 骨感染的临床问题，通过正式了解这一过程，确定新的药物靶点，以及 探索3D打印抗生素浸渍间隔物用于单期局部给药的潜力 翻修手术。

ABSTRACT Prosthetic joint infection (PJI), the vast majority of which is caused by Staphylococcal species, is the bane of elective total joint replacement surgery. The pathogenic mechanisms responsible for the unique problems of PJI, which render these infections incurable, remain largely unknown. To address this gap in knowledge, we performed extensive transmission electron microscopy (TEM) studies that uncovered novel, previously unreported mechanisms of S. aureus colonization of canaliculi and submicron cracks in cortical bone. Our novel observations suggest, counter to the well-accepted dogma, that S. aureus must have motility mechanisms that allow it to identify canaliculi and submicron cracks by geometric and rigidity cues from the 3D extracellular matrix of bone, and subsequently deform from spherical cocci into rod shaped bacteria that propel its mitotic progeny through asymmetric septal planes through the submicron canaliculi. This mechanism shelters the S. aureus in these submicron cracks and canaliculi such that leukocytes become incapable of reaching them. This also likely limits the effectiveness of antimicrobials and renders the infection incurable. Thus, our global hypothesis is that S. aureus utilizes haptotaxis and durotaxis (directional mobility guided by geometric and rigidity cues from the 3D extracellular matrix, respectively), to incurably colonize canaliculi and submicron channels in cortical bone. In this application, we take innovative genetic and small molecule screening approaches to design new generations of antimicrobials that inhibit haptotaxis- and durotaxis- mediated colonization of cortical bone. In Aim 1, we use innovative nanoporous silicon membrane transwell chambers to define kinetics of occupancy and migration of S. aureus through submicron channels ex vivo, to simulate in vivo haptotaxis and durotaxis through canaliculi. We also propose to complete a case-control clinical correlate study documenting S. aureus colonization of microcracks and osteocytic-canalicular networks of infected human cortical bone. In Aim 2, we take a focused candidate gene analysis and non-biased de novo genetic screens, with complementary empiric TnSeq mutant library screen approach to identify S. aureus genes involved in canalicular invasion and migration. In Aim 3, we propose to develop 3D-printed spacers infused with novel antibiotics, that target essential enzymes for RNA and protein synthesis in biofilm- associated bacteria or essential proteins involved in haptotaxis and durotaxis, to demonstrate the efficacy of single-stage revision of septic femoral plates in an established OM murine model. The multidisciplinary approach, encompassing tissue engineering and 3D printing, microbial genomics, and high throughput screening of small molecule antimicrobials, will provide critical information needed to solve the significant clinical problems of bone infection by formally understanding this process, identifying novel drug targets, and exploring the potential of localized delivery using 3D-printed antibiotic-impregnated spacers for single-stage revision surgery.