Zinc Homeostasis in Bacillus subtilis

描述（由申请人提供）：锌是所有细胞的必需营养素，因为其作为催化和结构辅因子的关键作用。对锌和其他营养金属离子的竞争对于决定宿主-病原体相互作用的结果至关重要。金属调节蛋白感测锌缺乏和过量，并且是细菌适应锌可用性变化所需的。在革兰氏阳性模式生物枯草芽孢杆菌中， Zur（锌摄取抑制因子）和CzrA的锌过量。当锌水平限制生长时，Zur抑制被解除，导致其增加锌摄取（由ZnuABC转运蛋白介导），通过置换小的核糖体相关锌蛋白（L31和L33）的锌动员，以及通过不需要锌的替代酶替换关键的锌依赖性酶（FolE）。在下一个项目期间，我们将研究锌感应的详细分子机制，并确定细胞中不稳定锌池的主要成分。初步结果产生了五个核心假设，构成了具体目标的基础。目的1a将解决的假设，Zur的调节涉及一个分级的响应，从锌充足的细胞过渡到缺乏，并将测试锌结合Zur在扩大锌响应范围的负协同作用。在目标1b中，我们将调整遗传编码的荧光报告基因，以监测体内游离锌水平，并寻求调和Zur和CzrA传感器的异常高（亚皮摩尔）锌亲和力与新兴证据一致，游离锌水平在中等皮摩尔范围内。目的2将讨论低分子量硫醇杆菌硫醇（BSH）作为主要缓冲剂的作用。 不稳定的锌库（Aim 2a），小的核糖体相关蛋白作为锌的主要储存形式的作用（Aim 2b），以及这两种锌库在FolE（一种对锌有效性敏感的锌金属酶）和其他关键锌酶的代谢中的作用。钙卫蛋白和其他可能的因素对锌的限制是人类先天免疫反应的关键部分。由于这里描述的所有关键锌稳态因子在许多重要的人类病原体中是保守的，这些研究将提供关键的背景信息，以促进影响金属离子稳态的抗菌剂的最终开发。

DESCRIPTION (provided by applicant): Zinc is an essential nutrient for all cells due to its key role as a catalytic and structural cofactor. Competition for zinc and other nutrient metal ions is critical for determining the outcome of host- pathogen interactions. Metalloregulatory proteins sense both zinc deficiency and excess and are required for bacterial acclimation to changes in zinc availability. In Bacillus subtilis, a Gram positive model organism, zinc sufficiency is sensed by Zur (zinc uptake repressor) and zinc excess by CzrA. When zinc levels are limiting for growth, Zur repression is relieved resulting it increased zinc uptake (mediated by the ZnuABC transporter), Zn mobilization by displacement of small, ribosome-associated zinc proteins (L31 and L33), and replacement of a key zinc dependent enzyme (FolE) by a non-zinc requiring alternate enzyme. In the next project period, we will investigate the detailed molecular mechanisms of zinc sensing and define the major components of the labile zinc pool in the cell. Preliminary results have led to five core hypotheses that form the foundation of the Specific Aims. Aim 1a will address the hypothesis that regulation by Zur involves a graded response as the cell transitions from zinc sufficiency to deficiency, and will test the role of negative cooperativity in zinc-binding to Zur in expanding the range of zinc responsiveness. In Aim 1b, we will adapt genetically encoded fluorescent reporters to monitor free zinc levels in vivo and seek to reconcile the exceptionally high (sub-picomolar) zinc affinity of the Zur and CzrA sensors with emerging evidence consistent with free zinc levels in the mid-picomolar range. Aim 2 will address the role of the low molecular weight thiol bacillithiol (BSH) as the major buffer of the labile zinc pool (Aim 2a), the role of small, dispensable ribosome-associated proteins as the major storage form of zinc (Aim 2b), and the roles of these two zinc pools in metallation of FolE, a zinc metalloenzyme sensitive to zinc availability, and other key zinc enzymes. Limitation for zinc by calprotectin, and likely other factors, is a key part of the human innate immune response. Since all of the key zinc homeostasis factors described here are conserved in many important human pathogens, these studies will provide key background information to facilitate the ultimate development of antimicrobial agents that affect metal ion homeostasis.