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

项目 1：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: 10247795
• 负责人: Hani A Awad
• 金额: $ 15.33万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10247795
• 关键词: 3-Dimensional; 3D Print; Address; American; Amputation; Antibiotics; Bacteria; Biological Assay; Bone Matrix; Caliber; Candidate Disease Gene; Cells; Cessation of life; Chronic; Clinical; Cues; Custom; Debridement; Effectiveness; Enzymes; European; Extracellular Matrix; Foreign Bodies; Generations; Genes; Genetic; Genetic Screening; Genus staphylococcus; Human; Image; In Vitro; Infection; Intervention; Joint Prosthesis; Kinetics; Knowledge; Lead; Leukocytes; Libraries; Mediating; Membrane; Microbial Biofilms; Mitotic; Modeling; Morphogenesis; Mus; Nanoporous; Nature; Operative Surgical Procedures; Organism; Orthopedics; Osteomyelitis; Osteotomy; Pathogenicity; Patients; Phenotype; Process; Protein Biosynthesis; Proteins; RNA chemical synthesis; Replacement Arthroplasty; Rod; Safety; Shelter facility; Side; Silicon; Staphylococcus aureus; Staphylococcus aureus infection; Testing; Tissue Engineering; Translational Research; Transmission Electron Microscopy; antimicrobial; base; bone; case control; cell motility; combinatorial; confocal imaging; cortical bone; daughter cell; design; high throughput screening; in vivo; inhibitor/antagonist; innovation; interdisciplinary approach; joint infection; lead candidate; methicillin resistant Staphylococcus aureus; microbial genomics; migration; mouse model; mutant; nanopore; new therapeutic target; novel; novel therapeutics; osteoimmunology; screening; septic; small molecule; soft tissue; submicron; transposon sequencing

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.

摘要 人工关节感染（PJI），其中绝大多数是由葡萄球菌引起的， 择期全关节置换手术致病机制负责的独特问题， PJI使这些感染无法治愈，在很大程度上仍然未知。为了填补这一知识空白，我们 进行了广泛的透射电子显微镜（TEM）研究，发现了新的，以前 未报道的S.骨皮质中小管和亚微米裂纹的金黄色葡萄球菌定植。我们 新的观察表明，反对广为接受的教条，S。金黄色葡萄球菌必须具有运动性 机制，使其能够识别小管和亚微米裂纹的几何和刚性线索，从三维 骨细胞外基质，随后从球形球菌变形为杆状细菌， 其有丝分裂后代通过亚微小管穿过不对称的间隔平面。这一机制 庇护着S.金黄色葡萄球菌在这些亚微米裂缝和小管，使白细胞变得无法 到达他们。这也可能限制抗菌药物的有效性，并使感染无法治愈。 因此，我们的总体假设是S。aureus利用触觉和硬旋转（由 分别来自3D细胞外基质的几何和刚性线索），以不可治愈地定殖小管， 皮质骨中的亚微米通道在这个应用中，我们将创新的基因和小分子 筛选方法，以设计新一代的抗菌剂，抑制haptotaxis-和durotaxis- 介导的皮质骨定植。在目标1中，我们使用创新的纳米多孔硅膜transwell 室，以确定S.金黄色葡萄球菌通过离体亚微米通道， 模拟体内通过小管的触移和硬脊膜扩张。我们还建议完成一个病例对照 临床相关研究记录了S.微裂纹和骨细胞-小管网络的金黄色葡萄球菌定植 感染的人类皮质骨在目标2中，我们采取集中的候选基因分析和无偏见的从头 遗传筛选，用互补的经验性TnSeq突变体文库筛选方法鉴定S.金黄色 参与小管侵入和迁移的基因。在目标3中，我们建议开发3D打印间隔物 注入新型抗生素，靶向生物膜中RNA和蛋白质合成的必需酶- 相关细菌或参与趋触性和硬骨扩张的必需蛋白质，以证明 在已建立的OM小鼠模型中对脓毒症股骨接骨板进行一期翻修。多学科 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。 通过正式了解这一过程、确定新的药物靶点，解决骨感染的临床问题，并 探索使用3D打印的植入物浸渍间隔物进行单阶段局部递送的潜力 翻修手术