Novel Catabolite Repression Pathway Controls Virulence in Streptococcus pyogenes

描述(申请人提供)：在体外已确定了许多影响化脓性链球菌毒力基因调控的调控基因和环境线索。然而，关于这些信号是如何被感知的，不同的调控通路是如何相互作用的，以及这些信号在体内是否相关，目前还知之甚少。这些都是重要的问题，因为对间隔特定线索的不同调控可能决定感染是进行自我限制还是组织破坏性结果，这一问题对于化脓性链球菌来说理解得很少。为了深入了解这些问题，我的实验室专注于SpeB的调控，SpeB编码分泌的SpeB半胱氨酸蛋白酶。我们定义了一套反映体内表达模式的体外条件，包括生长阶段、pH、氯离子浓度和富含碳水化合物/多肽的营养环境。通过突变来确定一个可以协调SpeB调控的调节因子来响应这些信号，导致了LacD.1的发现，LacD.1是一种抑制SpeB转录的标记糖醛缩酶。有趣的是，化脓性链球菌和其他几种革兰氏阳性病原菌含有两个乳糖操纵子(Lac.1和Lac.2)，其中分解代谢途径中LacD.1上游的几个编码酶的基因缺失或为假基因，表明Lac.2参与分解代谢，Lac.1已进化为调节功能。与此一致的是，LacD.2没有调节表型，不能补充LacD.1的调节表型，并且破坏LacD.1的催化中心的突变不会改变其调节功能；然而，其他可能改变其结合底物的能力的突变确实会破坏调节。此外，我们还证明了LacD.1在体内与RopB形成复合体，RopB是一种DMA结合蛋白，也是一种已知的转录调节基因和其他代谢基因。RopB是在革兰氏阳性病原体中广泛分布的转录调控因子RGG-家族的成员，对这一重要的调控因子家族如何与信号转导系统相互作用几乎一无所知。根据其他醛缩酶和糖分解代谢酶如何适应调节功能的例子，这些数据提出了LacD.1功能的以下模型：1.LacD.1已被适应作为中间代谢的传感器；2.在富含碳水化合物的条件下，结合其底物使LacD.1充当“抗激活剂”并以非活性形式隔离RopB；3.LacD.1可能在碳分解代谢抑制和毒力基因表达中发挥更广泛的作用；以及4.LacD.1对毒力是重要的。本项目将探讨这些问题

DESCRIPTION (provided by applicant): Numerous regulatory genes and environmental cues that influence Streptococcus pyogenes virulence gene regulation in vitro have been identified. However, very little is currently known as to how these signals are sensed, how different regulatory pathways interact and whether these cues are relevant in vivo. These are important questions, as differential regulation in response to compartment-specific cues may determine whether infection proceeds to a self-limiting or tissue-destructive outcome, an issue that is only very poorly understood for S. pyogenes. To gain insight into these questions, my lab has focused on regulation of speB, which encodes the secreted SpeB cysteine protease. We defined a set of in vitro conditions, including growth phase, pH, Cl- anion concentration and a carbohydrate-poor/peptide rich nutritional environment, that reflects expression patterns measured in vivo. Mutagenesis to identify a regulatory factor that could coordinate speB regulation in response to each of these cues resulted in the discovery of LacD.1, a tagatose aldolase that acts to repress speB transcription. Interestingly, S. pyogenes and several other Gram-positive pathogens contain two lactose operons (Lac.1 and Lac.2) where several of the genes encoding enzymes upstream of LacD.1, but not LacD.2, in the catabolic pathway are missing or are pseudogenes, suggesting that Lac.2 is involved in catabolism and that Lac.1 has evolved to a regulatory function. Consistent with this, LacD.2 has no regulatory phenotype and cannot complement the regulatory phenotype of LacD.1 and mutations that disrupt the catalytic center of LacD.1 do not alter its regulatory function; however, other mutations that may alter its ability to bind substrate do ablate regulation. Furthermore, we have shown that LacD.1 forms a complex in vivo with RopB, a DMA-binding protein and a known regulator of transcription and other metabolic genes. RopB is a member of the Rgg-family of transcription regulators broadly distributed among Gram-positive pathogens and virtually nothing is known about how this important family of regulators interacts with signal transduction systems. Based on examples of how other aldolases and sugar catabolic enzymes have been adapted to regulatory functions, these data suggest the following model for LacD.1 function: 1. That LacD.1 has been adapted as a sensor of intermediary metabolism; 2. That under carbohydrate-rich conditions, binding its substrate allows LacD.1 to act as an "anti-activator" and sequester RopB in an inactive form; 3. That LacD.1 may play a broader role in carbon catabolite repression and virulence gene expression; and 4. that LacD.1 is important for virulence. This project will explore these questions