Novel Catabolite Repression Pathway Controls Virulence in Streptococcus pyogenes

新型分解代谢物抑制途径控制化脓性链球菌的毒力

• 批准号: 8084162
• 负责人: Michael G. Caparon
• 金额: $ 32.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8084162
• 关键词: Aldehyde-Lyases; Anions; Bacterial Infections; Binding; Binding Proteins; Biochemical; Carbohydrates; Carbon; Catabolism; Chimera organism; Complement; Complex; Cues; Culture Media; Cysteine Protease; Data; Dominant-Negative Mutation; Environment; Enzymes; Evolution; Family; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Gram-Positive Bacteria; Growth; Human; In Vitro; Infection; Lac Operon; Lactose; Maps; Measures; Metabolic; Metabolism; Modeling; Molecular Profiling; Mus; Mutagenesis; Mutation; Nutritional; Outcome; Pathogenesis; Pathway interactions; Pattern; Peptides; Phase; Phenotype; Physiological; Play; Pseudogenes; Regulation; Regulator Genes; Regulatory Pathway; Repression; Research Personnel; Role; Signal Transduction; Specificity; Streptococcus pyogenes; Structure; Testing; Tissues; Transcription Coactivator; Virulence; Virulence Factors; base; in vivo; insight; member; mutant; novel; pathogen; programs; response; sensor; subcutaneous; sugar; tagatose

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

描述（由申请方提供）：已鉴定出影响体外化脓性链球菌毒力基因调控的许多调控基因和环境因子。然而，目前很少有人知道这些信号是如何被感知的，不同的调节途径如何相互作用，以及这些信号是否与体内有关。这些都是重要的问题，因为对特定区室线索的差异调节可能决定感染是否会导致自限性或组织破坏性结果，这是一个对S.化脓为了深入了解这些问题，我的实验室专注于speB的调控，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贫乏/肽丰富的营养环境，其反映了体内测量的表达模式。通过诱变来鉴定一种调节因子，该调节因子可以响应于这些线索中的每一个来协调speB调节，从而发现了LacD.1，一种用于抑制speB转录的塔格糖醛缩酶。有趣的是，S.化脓性链球菌和几种其它革兰氏阳性病原体含有两个乳糖操纵子（Lac.1和Lac.2），其中在分解代谢途径中编码LacD.1上游酶的几个基因缺失或为假基因，但LacD.2不是，这表明Lac.2参与分解代谢，并且Lac.1已进化为调节功能。与此一致，LacD.2没有调节表型，不能补充LacD.1的调节表型，破坏LacD.1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能力的突变会消除调节。此外，我们已经表明，LacD.1在体内与RopB形成复合物，RopB是一种DMA结合蛋白，也是一种已知的转录调节因子和其他代谢基因。RopB是广泛分布在革兰氏阳性病原体中的Rgg转录调节因子家族的成员，但对于这个重要的调节因子家族如何与信号转导系统相互作用几乎一无所知。基于其他醛缩酶和糖分解代谢酶如何适应调节功能的实例，这些数据表明LacD.1功能的以下模型：1. LacD.1已被改编为中间代谢的传感器; 2.在富含碳水化合物的条件下，结合其底物允许LacD. 1充当“抗激活剂”并以非活性形式螯合RopB; 3. LacD.1可能在碳代谢产物阻遏和毒力基因表达中发挥更广泛的作用; 4. LacD.1对毒力很重要本项目将探讨这些问题