Cloning/Characterization of S. mutans Iron Stimulation

描述（由申请人提供）：铁是细菌病原体及其哺乳动物宿主必需的微量营养素，因此对这种元素的竞争是感染过程的一个组成部分。细胞外亚铁对入侵的病原体在生物学上是不可用的，因为它在很大程度上被隔离在宿主蛋白质上，并且在其自由形式下迅速氧化成不溶的铁态。细菌通常通过开启铁清除系统和毒力决定因素来响应宿主的铁保留系统，以促进细菌的生存和传播。在革兰氏阳性菌中，研究最广泛的金属离子依赖性调节因子是白喉杆状杆菌中的DtxR（一种dna结合蛋白），及其在表皮葡萄球菌中的系统发育同源物SirR和在金黄色葡萄球菌中的MntR。重要的是，这些金属调节因子作为转录因子调节细菌毒力基因的表达和与毒力和氧化应激相关的高亲和力金属离子摄取系统。我们的实验室对变形链球菌（人类牙菌斑的主要致酸成分）进行了研究，发现DtxR同源物可以调节一系列基因，并且越来越多的证据表明铁在变形链球菌生物膜形成、氧化应激反应和耐酸性中的作用是一致的。事实上，鉴定和表征参与这些和其他致病过程的基因是一个高度优先事项，因为它们可以证明是未来的药物靶点。本研究计划的主要目标将集中在阐明铁依赖基因调控及其在变形链球菌诱导龋齿发生中的作用。具体目的包括：1)表征属于假定的变形链球菌Dlg调控的铁反应基因；2)克隆和鉴定变形链球菌中其他不受Dlg控制的铁依赖性基因；3)检测变形链球菌Dlg和铁反应基因在近似人类口腔环境中的表达；4)建立Dlg/铁调控基因表达与变形链球菌诱导龋齿发生的相关性。

DESCRIPTION (provided by applicant): Iron is an essential micronutrient for bacterial pathogens and their mammalian hosts, and hence competition for this element is an integral component of the infectious process. Extracellular ferrous iron is biologically unavailable to invading pathogens because it is largely sequestered to host proteins, and in its free form rapidly oxidizes to the insoluble ferric state. Bacteria often respond to the iron withholding system of the host by turning on iron scavenging systems and virulence determinants to promote bacterial survival and dissemination. Among the most extensively studied metal ion-dependent regulators in Gram-positive bacteria is DtxR, a DNA-binding protein, in Corynebacterium diphtheriae, and its phylogenetic homologs SirR in Staphylococcus epidermidis and MntR in Staphylococcus aureus. Importantly, these metalloregulators function as transcription factors that modulate the expression of bacterial virulence genes and high affinity metal ion uptake systems related to virulence and oxidative stress. Work conducted in our laboratory on Streptococcus mutans, the principal acidogenic component of human dental plaque, revealed a DtxR homolog that regulates a repertoire of genes, and accumulating evidence is consistent with a role for iron in S. mutans biofilm formation, the oxidative stress response, and acid tolerance. Indeed, the identification and characterization of genes involved in these and other pathogenic processes is a high priority since they can prove to be future drug targets. The major goal of this research proposal will focus on elucidating iron-dependent gene regulation and its role in S. mutans-induced cariogenesis. The specific aims include 1) characterizing iron-responsive genes that belong to a putative S. mutans Dlg regulon; 2) cloning and characterizing other iron-dependent genes in S.mutans that are not subject to Dlg control; 3) examining the expression of S. mutans Dlg- and iron-responsive genes in environments that approximate the human oral cavity; and 4) establishing a correlation between Dlg/iron-regulated gene expression and S. mutans-induced cariogenesis.