Determination of structure-function relationships and role in virulence of a MerR-type regulator that mediates zinc tolerance in Streptococcus mutans

摘要 龋齿是最常见的慢性传染病，估计治疗费用约为70美元 十亿/年。变形链球菌是一种多因素致病因素，是一种重要的龋病致病菌 因为它具有调节口腔生物膜的能力，从而促进高度 产酸(致龋齿)微生物区系。微量金属铁、锰和锌(锌)具有结构性、催化性、 以及蛋白质中的调节作用，因此对所有形式的生命都是必不可少的。相反，这些相同的金属 过量时有毒，因此维持金属动态平衡的能力是寄主病原体的关键方面 互动。锌的毒性来自于它在欧文-威廉姆斯系列金属稳定性中的最高地位，这 允许锌贪婪地与非同源金属蛋白结合，使它们失去功能。因为它两者都有 抗菌和免疫调节特性以及对哺乳动物细胞的毒性相对较低，锌已被 在医学上使用了几十年，包括将其加入口腔保健品中治疗牙龈炎， 口臭，防止结石形成。最近发现，变形链球菌在本质上是一种 比所有其他链球菌对锌的毒性水平有更高的耐受性，这种高耐受性是 由一个被命名为ZccE的独特的P型ATPase输出子介导。ZccE的表达受 一种独特的Merr型调节器，ZccR代表ZccE调节器，它强烈激活ZccE转录，以响应 高锌胁迫。因为ZccE和ZccR都是变形链球菌独有的，Lemos实验室认为两者都可以 作为开发针对变形链球菌的锌为基础的疗法的目标。而其他项目则在 Lemos实验室正在探索ZccE的抗菌潜力，该应用中心的具体目标是围绕 ZccR监管机构。本应用程序的目标是确定ZccR的结构-功能关系 并探索其作为抗菌靶标的潜力。为了实现这些目标，PI将(I)使用 结晶学和基于计算的方法来确定ZccR的结构并确定其临界 (Ii)利用大鼠模型确定ZccR对口腔生物膜的贡献 单独或联合局部锌治疗的定植和龋齿发展。从以下方面获得的知识 这项研究将有助于ZccR小分子抑制剂的结构指导设计，并揭示ZccR的 锌稳态靶向机制对抗变形链球菌感染的潜力，与长期 目的开发预防龋病和治疗系统性变形链球菌的新疗法 感染(即感染性心内膜炎)。这项工作将在高度支持和协作的情况下进行 研究环境，目标1在Lemos和McKenna实验室之间建立合作。 此外，提供的全面培训计划将促进PI的学术和科学成长 通过参加正式课程工作和讲习班，有机会学习新的方法， 指导学生，建立新的合作关系，提高科学写作和演示技能。

Abstract Dental caries is the most prevalent chronic infectious disease, with an estimated treatment cost of ~$70 billion/year. While a multifactorial disease, Streptococcus mutans is recognized as a keystone caries pathogen because of its capacity to modulate the oral biofilm in a way that promotes the establishment of a highly acidogenic (cariogenic) microbiota. The trace metals iron, manganese, and zinc (Zn) play structural, catalytic, and regulatory roles in proteins and so are essential to all forms of life. Conversely, these same metals are toxic when in excess such that the ability to maintain metal homeostasis is a critical aspect of host-pathogen interactions. The toxicity of Zn derives from its top position in the Irving-Williams series of metal stability, which allows Zn to avidly bind to non-cognate metalloproteins rendering them nonfunctional. Because it has both antimicrobial and immunomodulatory properties and relatively low toxicity to mammalian cells, Zn has been used for decades in medicine, including incorporation into oral health products for the treatment of gingivitis, halitosis, and prevention of calculus formation. Recently, it was discovered that S. mutans is intrinsically and substantially more tolerant to toxic levels of Zn than all other streptococci and that this high tolerance is mediated by a unique P-type ATPase exporter that has been named ZccE. Expression of zccE is controlled by a unique MerR-type regulator, ZccR for zccE regulator, that strongly activates zccE transcription in response to high Zn stress. Because both ZccE and ZccR are unique to S. mutans, the Lemos lab proposes that both can be targeted for the development of a Zn-based therapy tailored against S. mutans. While other projects in the Lemos lab are exploring the antimicrobial potential of ZccE, the specific aims of this application center around the ZccR regulator. The goals of this application are to determine the structure-function relationships of ZccR and explore its potential as an antimicrobial target. To accomplish these goals, the PI will (i) use crystallography and computational-based approaches to determine the structure of ZccR and identify its critical functional residues, and (ii) utilize the rat model to determine the contribution of ZccR to oral biofilm colonization and caries development alone or in combination with topical Zn treatment. Knowledge gained from this study will facilitate the structure-guided design of small molecule inhibitors for ZccR and reveal the potential of targeting mechanisms of Zn homeostasis to combat S. mutans infections, with the long-term objective of developing novel therapies for the prevention of dental caries and treatment of systemic S. mutans infections (i.e., infective endocarditis). This work will be conducted in a highly supportive and collaborative research environment with Aim 1 establishing a collaboration between the Lemos and McKenna labs. Moreover, the comprehensive training plan provided will accelerate the PI's academic and scientific growth through participation in formal course work and workshops, opportunities to learn new methodologies, mentoring students, establishing new collaborations, and improving scientific writing and presentation skills.