The impact of hypoxia on Staphylococcus aureus metabolism and virulence during osteomyelitis

项目总结/摘要 金黄色葡萄球菌是一种重要的人类病原体，能够引起危及生命的感染 在多种宿主组织中。骨髓炎是一种侵袭性和使人衰弱的骨感染， 葡萄球菌病的常见表现。事实上，骨髓炎约占2-3的 在美国，每1,000例儿科医院的入院病例中， 骨折众所周知，骨感染由于广泛的抗菌素耐药性而难以治疗 和病原体诱导的骨重建，这限制了抗生素渗透到感染病灶。S. 金黄色葡萄球菌是迄今为止最常见的原因肌肉骨骼感染，但机制， 葡萄球菌在骨内存活并最终破坏骨是知之甚少的。的总体目标 这个建议是为了了解S.金黄色葡萄球菌调节其毒力和代谢程序，以在 骨髓炎期间的骨骼。 骨骼，像大多数哺乳动物组织一样，天生就是缺氧的。此外，维持骨骼健康 需要通过常驻的骨形成成骨细胞和骨再吸收破骨细胞进行恒定的骨更新。这些 反过来，骨骼细胞需要以高葡萄糖摄取率为特征的专门代谢， 预计将限制入侵病原体的碳源。为了更好地理解S。 金黄色葡萄球菌在这种缺氧和代谢独特的环境中茁壮成长，我们创造了一个强大的小鼠模型， 能够精确定量细菌负荷和骨转换。通过应用 转座子测序（TnSeq），我们确定了超过200个葡萄球菌基因 对骨骼中的生存很重要。重要的是，细菌对缺氧的反应被发现是至关重要的。 生存的决定因素在骨髓炎，缺氧生长不仅决定了能源生产 葡萄球菌使用的策略，而且还增加了群体依赖性毒力因子的产生， 参与骨质破坏。基于这些初步数据，我们假设S。金黄色葡萄球菌存活率 骨通过（a）应答缺氧的群体调节的毒力因子表达和（B）特异性 营养利用计划，使骨骼在独特的代谢环境中生长。拟议 目的将测试这一假设，以确定（i）缺氧生长触发增加的机制， 葡萄球菌毒力，（ii）支持细菌在骨中生长的代谢途径，以及（iii） 宿主缺氧信号通路在骨髓炎抗菌免疫和骨重建中的作用 这些研究的完成将阐明侵入性感染期间微生物的生存策略，确定 缺氧对细菌发病机制影响，并有助于满足新发骨髓炎的迫切需要 通过定义假定的抗微生物和抗毒力靶点来研究治疗方法。

PROJECT SUMMARY / ABSTRACT Staphylococcus aureus is an important human pathogen, capable of causing life-threatening infections in a variety of host tissues. Osteomyelitis, an invasive and debilitating infection of bone, is one of the most common manifestations of staphylococcal disease. Indeed, osteomyelitis accounts for approximately 2-3 of every 1,000 admissions to pediatric hospitals in the United States, and complicates up to 25% of open fractures. Bone infections are notoriously refractory to treatment due to widespread antimicrobial resistance and pathogen-induced bone remodeling, which limits penetration of antibiotics into the infectious focus. S. aureus is by far the most common cause of musculoskeletal infection, yet the mechanisms by which staphylococci survive within and ultimately destroy bone are poorly understood. The overarching objective of this proposal is to understand how S. aureus regulates its virulence and metabolic programs to survive within bone during osteomyelitis. Bone, like most mammalian tissues, is inherently hypoxic. Moreover, maintenance of skeletal health requires constant bone turnover by resident bone-forming osteoblasts and bone-resorbing osteoclasts. These skeletal cells, in turn, require a specialized metabolism characterized by high rates of glucose uptake, which is expected to limit the carbon sources available to invading pathogens. In order to better understand how S. aureus thrives within this hypoxic and metabolically unique environment, we created a powerful murine model of osteomyelitis capable of precise quantification of both bacterial burdens and bone turnover. By applying transposon sequencing (TnSeq) to this osteomyelitis model, we identified >200 staphylococcal genes important for survival in bone. Importantly, bacterial responses to hypoxia were found to be critical determinants of survival during osteomyelitis, as hypoxic growth not only dictates the energy production strategies used by staphylococci, but also augments the production of quorum-dependent virulence factors that participate in bone destruction. Based on these preliminary data, we hypothesize that S. aureus survival in bone is facilitated by (a) quorum-regulated virulence factor expression in response to hypoxia, and (b) specific nutrient utilization programs that enable growth in the unique metabolic environment of bone. The proposed Aims will test this hypothesis to determine (i) the mechanism by which hypoxic growth triggers increased staphylococcal virulence, (ii) the metabolic pathways that support bacterial growth in bone, and (iii) the role of host hypoxic signaling pathways in antibacterial immunity and bone remodeling during osteomyelitis. Completion of these studies will elucidate microbial survival strategies during invasive infection, determine the impact of hypoxia on bacterial pathogenesis, and help to meet a critical need for new osteomyelitis therapeutics by defining putative antimicrobial and anti-virulence targets.