Zinc Homeostasis in Bacillus subtilis

说明(申请人提供)：锌是所有细胞的必需营养素，因为它是催化和结构辅因子的关键角色。对锌和其他营养金属离子的竞争对于决定宿主-病原菌相互作用的结果至关重要。金属调节蛋白既可以感知锌的缺乏，也可以感觉到锌的过剩，是细菌适应锌供应变化所必需的。在革兰氏阳性模式生物枯草芽孢杆菌中，锌是充足的 由Zur(锌吸收抑制因子)和ZrA过量的锌。当锌水平限制生长时，Zur抑制被缓解，导致锌摄取增加(由ZnuABC转运蛋白介导)，通过置换与核糖体相关的小锌蛋白(L31和L33)来动员锌，并将关键的锌依赖酶(FOE)替换为非锌需要的替代酶。在接下来的项目期间，我们将研究锌感应的详细分子机制，并确定细胞内不稳定锌库的主要成分。初步结果导致了五个核心假设，这些假设构成了具体目标的基础。目标1a将解决这一假设，即Zur的调节涉及细胞从锌充足到缺乏的分级反应，并将测试负协同作用在锌与Zur结合中的作用，以扩大锌的响应范围。在目标1b中，我们将采用基因编码的荧光记者来监测体内的游离锌水平，并寻求将Zur和ChurA传感器极高的(亚皮摩尔)锌亲和力与新出现的证据相一致，这些证据与中等皮摩尔范围内的游离锌水平一致。目标2将阐述低分子硫醇杆菌硫醇(BSH)作为主要缓冲剂的作用 不稳定的锌库(Aim 2a)，作为锌的主要储存形式的小的、不必要的核糖体相关蛋白的作用(Aim 2b)，以及这两个锌库在Fole金属化中的作用，Fole是一种对锌的有效性敏感的锌金属酶，以及其他关键的锌酶。钙保护素和可能的其他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的锌稳态因子在许多重要的人类病原体中都是保守的，这些研究将为最终开发影响金属离子稳态的抗菌剂提供关键的背景信息。

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.