Transcriptional Regulation by a Deacetylase, CobB, in a Periodontopathogen

牙周病原菌中脱乙酰酶 CobB 的转录调节

• 批准号: 8450690
• 负责人: DAVID J KOLODRUBETZ
• 金额: $ 17.94万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8450690
• 关键词: Acetate-CoA Ligase; Acetates; Acetylation; Acetylesterase; Adult; Affect; Antibodies; Arginine; Bacteria; Bacterial RNA; Binding; Biological; Cardiovascular Diseases; Cell physiology; Cells; Chemotaxis; Codon Nucleotides; Coupled; DNA-Binding Proteins; Deacetylase; Development; Disease; Enzymes; Escherichia coli; Eukaryota; Future; Genes; Genetic Transcription; Glutamine; Goals; Gram-Negative Bacteria; Growth; Homologous Gene; Infection; Inflammatory; Lead; Life; Lysine; Mass Spectrum Analysis; Metabolic; Metabolism; Microarray Analysis; Molecular Profiling; Organism; Pathway interactions; Pattern; Peptides; Periodontal Diseases; Periodontal Pocket; Periodontitis; Phase; Phosphorylation; Physiological; Physiology; Play; Protein Family; Proteins; Protocols documentation; RNA; Regulation; Regulatory Pathway; Regulon; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Risk Factors; Role; Salmonella enterica; Shapes; Signal Transduction; Sir2-like Deacetylases; Site; Systemic infection; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Tooth Loss; Transcription Regulatory Protein; Transcriptional Regulation; Virulence; Virulence Factors; cell type; enzyme activity; in vivo; interest; member; mutant; novel; pathogen; periodontopathogen; response; sensor; small molecule; soft tissue; transcription factor

DESCRIPTION (provided by applicant): Periodontitis is a disease of the subgingival crevice that leads to soft tissue degeneration and tooth loss. The bacterium Aggregatibacter actinomycetemcomitans (Aa) has been implicated in localized aggressive periodontitis, in several adult periodontal disorders and in life-threatening systemic infections. We are interested in determining how Aa is a successful periodontal pathogen. Our efforts have focused on defining novel regulatory pathways in Aa that accurately control expression of the virulence factors needed to thrive in different conditions, with the long term goal of developing therapeutic protocols to block the synthesis of coordinately regulated virulence proteins. Members of the sirtuin (Sir2-like) family of proteins are found in all three domains of life. These enzymes, which are NAD+-dependent protein deacetylases, play an important role in eukaryotic and archaeal transcriptional regulation by removing acetyl groups from lysines on various DNA-binding proteins. The bacterial Sir2 homologue is called CobB. In Salmonella enterica, this deacetylase has been shown to regulate the enzymatic activity of several enzymes in intermediary metabolism. Recently, three groups determined that ~90 proteins, including two transcription factors, are acetylated in E. coli and that S. enterica has 191 acetylated proteins, including nine transcription factors. However, the effect of acetylation on the activities of the transcription proteins was not tested. Given that the Sir2 deacetylase is important in controlling eukaryotic and archaeal transcription and that transcription factors can be acetylated in bacteria, we postulated that lysine acetylation of transcriptional regulatory proteins could play an important role in shaping the RNA expression patterns in bacteria, something that has not been shown in any bacterium. Thus, we made a deletion mutant of the Aa cobB deacetylase gene. Microarray analysis of RNA from this mutant showed that the alteration of acetylation levels in Aa leads to the mis-regulation of forty-eight RNAs, suggesting, but not proving, that lysine-acetylation of transcription factors will be critical for transcriptional regulation in bacteria. In this proposal, we will test this hypothesis. First, mass spectrometry will be used to identify the Aa "acetylome", including its lysine-acetylated transcription factors. Subsequently, we will construct a set of site-specific mutants, which mimic an acetylated lysine or a deacetylated lysine, in the acetylated- lysine codon for each of the genes encoding any of the acetylated transcription factors found. Finally, microarray studies with the mutants will reveal the global RNA expression profiles of each "acetylated" mutant, and its corresponding "deacetylated" mutant, relative to wild type cells. Finding different patterns of transcription among these strains would prove, for the first time in any bacterium, that lysine-acetylation of transcriptional regulatory proteins plays a key role in modulating bacterial RNA expression. Importantly, since CobB is an NAD+-dependent deacetylase, our results would also suggest that there is a possible new connection between physiology (NAD levels) and transcriptional regulation (acetylation) in bacteria.

描述（由申请人提供）：牙周炎是一种导致软组织退化和牙齿脱落的龈下裂隙疾病。伴放线菌聚集杆菌（Aggregatibacteractinomycetemcomitans，Aa）与局部侵袭性牙周炎、几种成人牙周疾病和危及生命的全身性感染有关。我们有兴趣确定Aa是如何成功的牙周病原体。我们的努力集中在定义新的调控途径，准确地控制在不同条件下茁壮成长所需的毒力因子的表达，与开发治疗方案的长期目标，以阻止协同调节毒力蛋白的合成。sirtuin（sir 2-like）蛋白质家族的成员存在于生命的所有三个领域。这些酶，这是NAD+依赖的蛋白脱乙酰酶，在真核生物和古细菌的转录调控中发挥重要作用，通过去除乙酰基从赖氨酸上的各种DNA结合蛋白。细菌Sir 2同源物被称为CobB。在肠道沙门氏菌中，这种脱乙酰酶已被证明可以调节中间代谢中几种酶的酶活性。最近，三个研究小组确定了约90个蛋白质，包括两个转录因子，在大肠杆菌中被乙酰化。coli和S.肠杆菌有191个乙酰化蛋白质，包括9个转录因子。然而，没有测试乙酰化对转录蛋白活性的影响。考虑到Sir 2脱乙酰酶在控制真核生物和古细菌转录中很重要，并且转录因子可以在细菌中乙酰化，我们假设转录调节蛋白的赖氨酸乙酰化可以在塑造细菌中的RNA表达模式中发挥重要作用，这在任何细菌中都没有表现出来。因此，我们制作了Aa cobB脱乙酰酶基因的缺失突变体。来自该突变体的RNA的微阵列分析表明，Aa中乙酰化水平的改变导致48种RNA的错误调节，这表明但不能证明转录因子的赖氨酸乙酰化对细菌中的转录调节至关重要。在本提案中，我们将检验这一假设。首先，质谱法将用于鉴定Aa“乙酰组”，包括其赖氨酸乙酰化的转录因子。随后，我们将构建一组位点特异性突变体，其模拟乙酰化赖氨酸或脱乙酰化赖氨酸，在乙酰化赖氨酸密码子中编码所发现的任何乙酰化转录因子的每个基因。最后，对突变体的微阵列研究将揭示相对于野生型细胞的每个“乙酰化”突变体及其相应的“脱乙酰化”突变体的全局RNA表达谱。在这些菌株中发现不同的转录模式将首次在任何细菌中证明，转录调节蛋白的赖氨酸乙酰化在调节细菌RNA表达中起着关键作用。重要的是，由于CobB是一种NAD+依赖性脱乙酰酶，我们的研究结果还表明，细菌中生理学（NAD水平）和转录调控（乙酰化）之间可能存在新的联系。