Lysine Acetylation in N. gonorrhoeae Quorum Sensing and Biofilm Formation

淋病奈瑟菌群体感应和生物膜形成中的赖氨酸乙酰化

• 批准号: 8705141
• 负责人: Michael A. Apicella
• 金额: $ 83.12万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-05 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8705141
• 关键词: Acetylation; Acetyltransferase; Affect; Antibiotic Therapy; Antimicrobial Resistance; Azithromycin; Bioinformatics; Biology; Bulla; Ceftriaxone; Cell division; Cell surface; Centers for Disease Control and Prevention (U.S.); Cervical; Cervix Uteri; Chromosomes; Collaborations; DNA; Data; Deacetylase; Development; Emergency Situation; Environment; Epithelial Cells; Exhibits; Gene Expression; Gene Expression Profile; Gene Proteins; Genes; Genetic Transcription; Gonorrhea; Grant; Growth; Health; Histone Deacetylase; Human; Human Papillomavirus; Immune; In Vitro; Infection; Laboratories; Lysine; Mass Spectrum Analysis; Membrane; Messenger RNA; Microbial Biofilms; Mind; Mutation; Neisseria gonorrhoeae; Nitric Oxide; Operon; Oral; Organism; Pathogenesis; Pathway interactions; Peptides; Peptidoglycan; Pharmaceutical Preparations; Phosphotransferases; Play; Post-Translational Protein Processing; Protein Family; Proteins; Proteome; Regulator Genes; Regulon; Reporting; Resistance; Role; Signal Transduction; Surface; System; Techniques; Testing; base; design; effective therapy; enzyme biosynthesis; mutant; novel; nuclease; pathogen; public health relevance; quorum sensing; respiratory; response; sensor; vaccine development

DESCRIPTION (provided by applicant): The Apicella, Gibson, Kirby and Wolfe labs have made the unique observations that Neisseria gonorrhoeae proteins undergo extensive N?-lysine acetylation and that quorum sensing coordinates this post-translational modification. Our studies have also shown that the degree of N-lysine acetylation effects survival in biofilms. Utilizing novel mass spectrometry techniques, Dr. Gibson's lab has shown that mutants of the luxS operon and ackA exhibit a significant increase in acetylated proteins. One such acetylated protein is the response regulator, MisR, which partners with its cognate sensor kinase MisS to form a two-component signal transduction system (TCS). Our data strongly suggest that MisRS senses the LuxS product. Our preliminary results suggest that the luxS operon functions as a classic quorum sensing system through MisRS and that this TCS controls genes that modulate cell division and N?-lysine acetylation. qRT-PCR studies with Ng grown in broth cultures and continuous flow biofilms demonstrate that the product of luxS significantly influences the transcription of a number of genes important in biofilm formation, including the gonococcal nuclease that remodels biofilm matrix, cell division proteins (minD and ftsZ), peptidoglycan biosynthesis enzymes (murD-murF), regulatory genes (metR), a SIR-2 family protein deacetylase and misRS. The hypothesis guiding this proposal is that quorum sensing and acetylation coordinately regulate biofilm formation to affect pathogenesis. To test this hypothesis, we propose the following 3 specific aims: 1) to determine the transcriptome of key Ng genes in the LuxS-regulated network of genes and the genes associated with reversible acetylation. These studies will be performed on mRNA isolated from organisms in the biofilm and planktonic states, 2) To understand the factors controlling Ng biofilm development, we will carry out comprehensive and targeted analyses designed to identify changes in the transcriptome, proteome and acetylome of Ng strain 1291 grown under defined in biofilms and planktonically and 3) Using N. gonorrhoeae grown over HPV E6/E7 transformed human cervical epithelial cells (HCEC), we will carry out an analogous study of the transcriptome, proteome and acetylome to interrogate the possible role of bacterial/eukaryotic signaling on N. gonorrhoeae biofilm development. These studies have the potential to open a new range of novel targets for antibiotics for the treatment of gonorrhea.

描述（由申请人提供）：Apicella、Gibson、Kirby 和 Wolfe 实验室进行了独特的观察，即淋病奈瑟氏球菌蛋白质经历了广泛的 Nα-赖氨酸乙酰化，并且群体感应协调了这种翻译后修饰。我们的研究还表明，N-赖氨酸乙酰化程度影响生物膜的存活。 Gibson 博士的实验室利用新颖的质谱技术表明，luxS 操纵子和 ackA 的突变体表现出乙酰化蛋白质的显着增加。其中一种乙酰化蛋白是反应调节因子 MisR，它与其同源传感器激酶 MisS 配合形成双组分信号转导系统 (TCS)。我们的数据强烈表明 MisRS 感知到了 LuxS 产品。我们的初步结果表明，luxS 操纵子通过 MisRS 作为经典的群体感应系统发挥作用，并且该 TCS 控制调节细胞分裂和 Nα-赖氨酸乙酰化的基因。对肉汤培养物和连续流动生物膜中生长的 Ng 进行的 qRT-PCR 研究表明，luxS 的产物显着影响生物膜形成中许多重要基因的转录，包括重塑生物膜基质的淋球菌核酸酶、细胞分裂蛋白（minD 和 ftsZ）、肽聚糖生物合成酶（murD-murF）、调节性酶 基因（metR）、SIR-2家族蛋白脱乙酰酶和misRS。指导该提议的假设是群体感应和乙酰化协调调节生物膜形成以影响发病机制。为了检验这一假设，我们提出以下 3 个具体目标：1）确定 LuxS 调控基因网络中关键 Ng 基因的转录组以及与可逆乙酰化相关的基因。这些研究将对从处于生物膜和浮游状态的生物中分离的 mRNA 进行，2) 为了了解控制 Ng 生物膜发育的因素，我们将进行全面和有针对性的分析，旨在识别在生物膜和浮游状态下生长的 Ng 菌株 1291 的转录组、蛋白质组和乙酰基组的变化，以及 3) 使用在 HPV 上生长的淋病奈瑟菌 E6/E7 转化的人宫颈上皮细胞 (HCEC)，我们将对转录组、蛋白质组和乙酰基组进行类似的研究，以探讨细菌/真核信号传导对淋病奈瑟菌生物膜发育的可能作用。这些研究有可能为治疗淋病的抗生素开辟一系列新的靶点。