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

基本信息

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
项目类别：
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

项目摘要

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）：牙周炎是导致软组织变性和牙齿脱落和牙齿脱落的疾病。细菌聚集放线菌细菌（AA）与局部侵袭性牙周炎，几种成人牙周病和威胁生命的全身感染有关。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AA中的新型调节途径上，该途径准确地控制了在不同条件下繁殖所需的毒力因子的表达，其长期目标是开发治疗方案以阻止协调调节的毒力蛋白的合成。在生命的所有三个领域中都发现了Sirtuin（Sir2状）蛋白质家族的成员。这些酶是NAD+依赖性蛋白脱乙酰基酶，通过去除各种DNA结合蛋白上赖氨酸的乙酰基通过去除真核和古细菌的转录调控中起着重要作用。细菌SiR2同源物称为COBB。在肠道沙门氏菌中，该脱乙酰基酶已被证明可以调节中介代谢中几种酶的酶促活性。最近，三个组确定约90种蛋白质，包括两个转录因子，在大肠杆菌中被乙酰化，而肠链球菌具有191个乙酰化蛋白质，包括9个转录因子。但是，乙酰化对转录蛋白活性的影响未测试。鉴于SiR2脱乙酰基酶在控制真核和古细菌转录方面很重要，并且可以在细菌中进行转录因子，因此我们假设转录调节蛋白的赖氨酸乙酰化对细菌中RNA表达模式的塑造可能起重要作用，而在任何细菌中都没有显示出任何细菌。因此，我们制作了AA COBB脱乙酰基酶基因的缺失突变体。对该突变体的RNA进行的微阵列分析表明，AA中乙酰化水平的改变会导致48个RNA的错误调节，这表明但没有证明转录因子的赖氨酸 - 乙酰化对细菌中转录调节的转录至关重要。在此提案中，我们将检验该假设。首先，质谱法将用于识别AA“乙酰基团”，包括其赖氨酸 - 乙酰化转录因子。随后，我们将在乙酰化赖氨酸密码子中构建一组位点特异性突变体，这些突变体模拟乙酰化赖氨酸或脱乙酰化赖氨酸，用于编码编码任何乙酰化转录因子的每个基因。最后，使用突变体的微阵列研究将揭示每个“乙酰化”突变体的全局RNA表达谱，并相对于野生型细胞，其相应的“脱乙酰化”突变体。在这些菌株中找到不同的转录模式将在任何细菌中首次证明，转录调节蛋白的赖氨酸 - 乙酰化在调节细菌RNA表达中起关键作用。重要的是，由于COBB是NAD+依赖性的脱乙酰基酶，因此我们的结果还表明，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。公共卫生相关性：牙周炎是尺寸缝隙中组织的炎症性疾病，与尺寸菌群向革兰氏阴性细菌的急剧转移有关。这种疾病的某些形式是由辅助细菌聚集的放线菌菌群菌群引起的，这也被认为是心血管疾病的危险因素。重要的是要表征这种生物体使用的新调节机制，例如响应生理变化的转录因子的乙酰化，因为这种途径可能是未来治疗干预的新靶标。