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: 8243316
• 负责人: DAVID J KOLODRUBETZ
• 金额: $ 22.36万
• 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 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8243316
• 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. PUBLIC HEALTH RELEVANCE: Periodontitis, an inflammatory disease of tissues in the subgingival crevice, is associated with a dramatic shift in the subgingival microflora towards gram negative bacteria. Certain forms of this disease are caused by the facultative bacterium Aggregatibacter actinomycetemcomitans which is also considered a risk factor in cardiovascular diseases. It is important to characterize new regulatory mechanisms used by this organism, such as acetylation of transcription factors in response to physiologic changes, since such pathways may serve as novel targets for future therapeutic intervention.

描述（由申请人提供）：牙周炎是一种牙龈下间隙疾病，导致软组织退化和牙齿脱落。放线菌聚集杆菌（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。我们感兴趣的是确定如何Aa是一个成功的牙周病原体。我们的工作重点是在Aa中定义新的调控途径，准确控制在不同条件下茁壮成长所需的毒力因子的表达，长期目标是开发治疗方案，以阻断协调调节的毒力蛋白的合成。sirtuin （sir2样）蛋白家族的成员存在于生命的所有三个领域。这些酶是依赖NAD+的蛋白质去乙酰化酶，通过去除各种dna结合蛋白上赖氨酸的乙酰基，在真核生物和古细菌的转录调节中发挥重要作用。细菌的Sir2同源物被称为CobB。在肠沙门氏菌中，这种去乙酰化酶已被证明在中间代谢中调节几种酶的酶活性。最近，三个研究组确定大肠杆菌中约有90种蛋白被乙酰化，其中包括两种转录因子，而肠球菌中有191种蛋白被乙酰化，其中包括9种转录因子。然而，没有测试乙酰化对转录蛋白活性的影响。考虑到Sir2去乙酰化酶在控制真核生物和古细菌转录中起重要作用，并且转录因子在细菌中可以乙酰化，我们假设赖氨酸乙酰化的转录调节蛋白可能在塑造细菌RNA表达模式中起重要作用，这在任何细菌中都没有显示出来。因此，我们制作了一个Aa cobB去乙酰化酶基因的缺失突变体。对该突变体RNA的芯片分析显示，Aa乙酰化水平的改变导致48种RNA的错误调控，这表明，但不能证明，转录因子的赖氨酸乙酰化对细菌的转录调控至关重要。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“乙酰化”，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去乙酰化赖氨酸，在乙酰化赖氨酸密码子中为每个基因编码任何发现的乙酰化转录因子。最后，对突变体的微阵列研究将揭示相对于野生型细胞，每个“乙酰化”突变体及其对应的“去乙酰化”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是一种依赖NAD+的去乙酰化酶，我们的研究结果还表明，细菌的生理（NAD水平）和转录调节（乙酰化）之间可能存在新的联系。