Novel Catabolite Repression Pathway Controls Virulence in Streptococcus pyogenes

描述（由申请人提供）：已经确定了许多影响化脓性链球菌毒力基因体外调控的调控基因和环境线索。然而，对于这些信号是如何被感知的，不同的调节途径是如何相互作用的，以及这些信号在体内是否相关，目前所知甚少。这些都是重要的问题，因为对区室特异性线索的不同调控可能决定感染是进入自我限制还是组织破坏的结果，这一问题对化脓性链球菌知之甚少。为了深入了解这些问题，我的实验室专注于对speB的调节，它编码分泌的speB半胱氨酸蛋白酶。我们定义了一组体外条件，包括生长期、pH、Cl-阴离子浓度和碳水化合物匮乏/肽丰富的营养环境，以反映体内测量的表达模式。通过诱变来确定一种调节因子，该因子可以协调speB对这些线索的调节，从而发现了LacD。一种抑制speB转录的塔格糖醛缩酶。有趣的是，化脓性葡萄球菌和其他几种革兰氏阳性病原体含有两个乳糖操纵子（lac1和lac2），其中几个基因编码LacD上游的酶。1，但不是LacD。2、在分解代谢途径中缺失或为假基因，提示lac2参与分解代谢，而lac1已进化为具有调节功能。与此一致，LacD。2无调节性表型，不能与LacD的调节性表型互补。1和破坏LacD催化中心的突变。不改变其调节功能；然而，其他可能改变其结合底物能力的突变确实削弱了调控作用。此外，我们已经证明了LacD。1在体内与dma结合蛋白RopB和已知的转录调控因子及其他代谢基因形成复合体。RopB是广泛分布于革兰氏阳性病原体的转录调节因子rg家族的一员，而对于这一重要的调节因子家族如何与信号转导系统相互作用，我们几乎一无所知。基于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的以下模型。1.功能：LacD。1已被用作中间代谢的传感器；2. 在富含碳水化合物的条件下，结合其底物允许LacD。1充当“抗活化剂”，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；和4。LacD。1对毒性很重要。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