Transcriptional Regulation by a Deacetylase, CobB, in a Periodontopathogen

描述(申请人提供)：牙周炎是一种会导致软组织退化和牙齿脱落的龈下缝隙疾病。伴生放线杆菌(Aggregatibacter Actinomcetcomitans，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感染有关。我们感兴趣的是如何确定AA是一种成功的牙周病原体。我们的努力集中在定义AA中新的调控途径，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来阻止协调调节的毒力蛋白的合成。Sirtuin(类似Sir2)蛋白家族的成员在生命的所有三个领域都有发现。这些酶是依赖于NAD+的蛋白去乙酰酶，通过去除各种DNA结合蛋白上赖氨酸上的乙酰基，在真核生物和古生物的转录调控中发挥重要作用。细菌Sir2的同源物被称为Cobb。在肠沙门氏菌中，这种脱乙酰酶已被证明在中间代谢中调节几种酶的活性。最近，三个小组确定了包括两个转录因子在内的~90个蛋白质在大肠杆菌中被乙酰化，而肠杆菌有191个乙酰化蛋白质，包括9个转录因子。然而，乙酰化对转录蛋白活性的影响并未得到测试。鉴于Sir2脱乙酰酶在控制真核生物和古生物转录中的重要作用，以及转录因子在细菌中可以乙酰化，我们推测转录调节蛋白的赖氨酸乙酰化可能在塑造细菌的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，我们获得了AA Cobb脱乙酰酶基因的缺失突变体。对该突变体RNA的微阵列分析表明，AA中乙酰化水平的变化导致了48个RNA的错误调控，这表明(但不是证明)转录因子的赖氨酸乙酰化将对细菌的转录调控起关键作用。在这项提议中，我们将检验这一假设。首先，将使用质谱学来鉴定AA“乙酰化组”，包括其赖氨酸-乙酰化的转录因子。随后，我们将构建一组定点突变，它模拟乙酰化赖氨酸密码子中的乙酰化赖氨酸或去乙酰化赖氨酸，用于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每个“乙酰化”突变体及其相应的“去乙酰化”突变体相对于野生型细胞的整体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表达方面发挥关键作用。重要的是，由于Cobb是一种依赖NAD+的脱乙酰酶，我们的结果也表明在细菌的生理(NAD水平)和转录调节(乙酰化)之间可能存在新的联系。

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.