Identifying the contribution of zinc limitation to antibiotic tolerance during S. aureus infection

摘要 金黄色葡萄球菌是导致许多慢性和复发性疾病的主要人类病原体。 感染.这些感染通常对治疗无效，每年导致约20，000人死亡 仅在美国。奇怪的是，在体外药敏试验中，从这些感染中分离的菌株经常 表现出对抗生素的完全敏感性，这表明宿主中存在的环境因素可能 影响S。金黄色葡萄球菌抗生素敏感性了解这些因素如何控制抗生素敏感性将有助于 提高了柠檬酸S.金黄色葡萄球菌感染，并减缓耐药性的演变。 我们以前已经表明，在主机内的外在应激，包括暴露于活性氧， 由宿主先天免疫系统产生的活性氧（ROS）无意中使S.金黄色 通过抑制S.金黄色葡萄球菌代谢活性。然而，ROS暴露不能完全 说明S的容忍状态。金黄色葡萄球菌在体内，这表明其他不明因素存在于 宿主降低抗生素对沙门氏菌效力。金黄色。在这里，我们提出，宿主免疫蛋白钙卫蛋白 诱导S.通过使病原体缺乏锌来杀死金黄色葡萄球菌。锌是一种重要的辅因子 这是许多细菌金属酶活性所必需的，这些酶进行主要的宿主过程。锌- 饥饿的S.金黄色葡萄球菌表现出DNA合成、转录和 翻译，并且这些过程代表了杀菌抗生素作用的主要靶标。主机中介 因此，锌螯合可能无意中使S.金黄色葡萄球菌通过减少 主要抗生素靶标的活性。总的来说，我们假设锌限制诱导了抗生素耐受性， 在S.金黄色葡萄球菌感染期间，通过饮食或免疫调节改变锌的可用性， 影响抗生素在宿主体内的效力。 在AIM 1中，我们将探讨靶点失活在驱动S。金黄色葡萄球菌抗生素耐药性直接 降低全球DNA复制和翻译速率，并测量对S.金黄色葡萄球菌抗生素 易感性然后，我们将评估宿主介导的锌螯合在诱导这一过程中的作用。 表型在AIM 2中，我们将进入S的小鼠模型。金黄色葡萄球菌败血症，以评估改变 生理锌可用性（通过饮食或遗传操作），以增强或抑制抗生素功效 对于S.金黄色。总之，我们希望我们的研究结果将有助于提高我们对SA抗生素的理解 敏感性，并阐明如何利用这些知识来解决目前无法解决的感染。

Abstract Staphylococcus aureus is a major human pathogen responsible for numerous chronic and relapsing infections. These infections often do not respond to treatment, leading to approximately 20,000 annual deaths in the US alone. Paradoxically, during in vitro susceptibility testing, isolates from these infections frequently exhibit full sensitivity to administered antibiotics, suggesting that environmental factors present in the host may influence S. aureus antibiotic susceptibility. Understanding how these factors control antibiotic susceptibility will improve the resolution of recalcitrant S. aureus infection, and slow the evolution of resistance. We have previously shown that extrinsic stressors within the host including exposure to reactive oxygen species (ROS) produced by the host innate immune system inadvertently render subpopulations of S. aureus tolerant to antibiotic killing by suppressing S. aureus metabolic activity. However, ROS exposure cannot fully account for the tolerant state of S. aureus in vivo, suggesting that other unidentified factors present within the host reduce antibiotic efficacy against S. aureus. Here we propose that the host immune protein calprotectin induces an antibiotic tolerant state in S. aureus by starving the pathogen of zinc. Zinc is an essential cofactor required for the activity of numerous bacterial metalloenzymes that carry out the major host processes. Zinc- starved populations of S. aureus demonstrate significantly reduced rates of DNA synthesis, transcription, and translation, and these processes represent the primary targets of bactericidal antibiotic action. Host-mediated zinc sequestration may therefore inadvertently render S. aureus tolerant to antibiotic killing by reducing the activity of major antibiotic targets. Overall, we hypothesize that zinc limitation induces an antibiotic tolerant state in S. aureus during infection and that altering zinc availability through diet or immune modulation will influence antibiotic efficacy within the host. In AIM1 we will probe the role of target inactivation in driving S. aureus antibiotic tolerance by directly reducing global DNA replication and translation rates, and measuring the impact on S. aureus antibiotic susceptibility. We will then assess the contribution of host-mediated zinc sequestration in inducing this phenotype. In AIM2 we will move into a mouse model of S. aureus sepsis to assess the relevance of altering physiological zinc availability (through diet or genetic manipulation) to enhance or suppress antibiotic efficacy against S. aureus. In all, we expect that our findings will help improve our understanding of SA antibiotic susceptibility, and elucidate how such knowledge can be exploited to resolve currently unresolvable infections.