Regulated cell death during biofilm development

细菌生理学已经在细胞生长的背景下被广泛研究，但是分子生物学研究还不多。 细菌经历细胞死亡和溶解的细节仍然是一个几乎完全的谜。越来越 最近的一系列证据表明，细菌细胞的死亡和溶解涉及活性的、遗传编码的 这些机制对复杂的发育过程至关重要，如孢子形成和生物膜形成。 金黄色葡萄球菌cid和Irg操纵子编码调节细菌死亡的新蛋白， 溶解CidA和LrgA蛋白被认为在结构和功能上与噬菌体相似， 编码holins和antiholins，这些基因在细菌中的普遍分布表明， 它们起着保守的生理作用。最近的研究表明， CidA介导的细胞溶解在生物膜的发展，但具体的代谢和环境线索， 在生物膜生长的背景下调节CID和/或RG介导的细胞死亡和裂解仍然不清楚。低- 氧生长和内源性一氧化氮（NO）的产生都被认为是细胞生长的调节因子， 死亡和其他细菌的生物膜中的扩散，但这些过程中涉及的分子机制 并没有得到很好的理解。初步数据表明，在低氧条件下生长和NO 都是调节cid和Irg表达的有效信号。scdA和NO-还原酶（nor）基因， 分别参与亚硝化应激反应和NO代谢，也位于靠近 与临床分离株UAMS-1中的lytSR-lrgAB接近。因此，这一提议的核心假设是， S.金黄色葡萄球菌生物膜生长是一个重要的发展 在控制Cid-/Lrg-介导的细胞死亡和裂解中的信号。该项目的具体目标是：1）研究 生物膜发育过程中向厌氧代谢的转变及其对cid和Irg表达的影响，2） 研究NO在生物膜形成过程中的作用，以及3）确定分子机制， LytSR介导的调节cid和Irg表达在生物膜生长过程中的作用。时空 生物膜内需氧和厌氧代谢的模式将使用荧光报告物来确定 基因融合到需氧和厌氧启动子，cid和Irg在这些确定的区域内的表达将 通过激光捕获显微切割显微镜（LCM）和实时RT-PCR测量。NO的作用 将使用荧光染料监测供体和清除剂在生物膜发育期间对细胞死亡的影响， 还将评估这些化合物对cid和Irg表达的影响。详细的分子 还将进行LytSR信号转导级联的表征，以阐明这一作用。 在I 0 W-O2生长和生物膜发育过程中的调节系统。总的来说，这些研究将揭示新的 深入了解生物膜发育过程中细胞死亡和裂解的分子控制。 相关性（见说明）：

Bacterial physiology has been extensively studied in the context of cell growth, but the molecular details by which bacteria undergo cell death and lysis have remained a near complete mystery. A growing body of recent evidence suggests that bacterial cell death and lysis involves active, genetically-encoded mechanisms that are critical to complex developmental processes such as sporulation and biofilm formation. The Staphylococcus aureus cid and Irg operons encode novel proteins that regulate bacterial death and lysis. CidA and LrgA proteins are proposed to be structurally and functionally similar to bacteriophage- encoded holins and antihollns, and the ubiquitous distribution of these genes among bacteria suggests that they play a conserved physiological role. Recent studies have demonstrated an important biological role for CidA-mediated cell lysis during biofilm development, but the specific metabolic and environmental cues that regulate cid and /rg-mediated cell death and lysis within the context of biofilm growth remain ill-defined. Low- oxygen growth and endogenous nitric oxide (NO) production have both been implicated as regulators of cell death and dispersal in biofilm of other bacteria, but the molecular mechanisms involved in these processes are not well understood. Preliminary data have suggested that growth under low oxygen conditions and NO are both potent signals that regulate cid and Irg expression. The scdA and NO-reductase {nor) genes, involved in the nitrosative stress response and NO metabolism, respectively, are also located in close proximity to lytSR-lrgAB in the clinical isolate UAMS-1. Thus, the central hypothesis of this proposal is that the transition into anaerobic metabolism during S. aureus biofilm growth is an important developmental signal in the control of Cid-/Lrg-mediated cell death and lysis. The specific aims of this project are 1) to study the transition to anaerobic metabolism during biofilm development and its effect on cid and Irg expression, 2) to examine the role of NO during biofilm development, and 3) to determine the molecular mechanism and role of LytSR-mediated regulation of cid and Irg expression during biofilm growth. Temporal and spatial patterns of aerobic and anaerobic metabolism within the biofilm will be determined using fluorescent reporter genes fused to aerobic and anaerobic promoters, and cid and Irg expression within these defined regions will be measured by laser capture microdissection microscopy (LCM) and real-time RT-PCR. The effect of NO donors and scavengers on cell death during biofilm development will be monitored using fluorescent dyes, and the effect of these compounds on cid and Irg expression will also be assessed. A detailed molecular characterization of the LytSR signal transduction cascade will also be performed to elucidate the role of this regulatory system during I0W-O2 growth and biofilm development. Collectively, these studies will reveal new insights into the molecular control of cell death and lysis during biofilm development. RELEVANCE (See Instructions):