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: 9222702
• 负责人: JOHN R KIRBY
• 金额: $ 83.94万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 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/9222702
• 关键词: Acetylation; Acetyltransferase; Affect; Anaerobic Bacteria; 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 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 Analysis; Pathway interactions; Peptides; Peptidoglycan; Phenotype; Phosphotransferases; Play; Post-Translational Protein Processing; Protein Family; Proteins; Proteome; Quantitative Reverse Transcriptase PCR; Regulator Genes; Regulon; Reporting; Resistance; Role; Signal Transduction; Surface; System; Techniques; Testing; cost; design; effective therapy; enzyme biosynthesis; mutant; novel; novel therapeutics; nuclease; pathogen; public health relevance; quorum sensing; respiratory; response; sensor; transcriptome; vaccine development

DESCRIPTION (provided by applicant): The Apicella, Gibson, Kirby and Wolfe labs have made the unique observations that Neisseria gonorrhoeae proteins undergo extensive Nϵ-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 Nϵ-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，发现淋球菌蛋白质经历了广泛的Nϵ-赖氨酸乙酰化，并且群体感应协调了这一翻译后修饰。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率。利用新的质谱学技术，吉布森博士的实验室已经表明，LuxS操纵子和ackA的突变显示出乙酰化蛋白质的显著增加。一种这样的乙酰化蛋白质是反应调节蛋白MISR，它与其同源传感器激酶MISH合作形成一个两组分的信号转导系统(TCS)。我们的数据有力地表明，MisRS感觉到了LuxS产品。我们的初步结果表明，LUXS操纵子通过MISRS起着经典的群体感应系统的作用，这种操纵子控制着调控细胞分裂和Nϵ-赖氨酸乙酰化的基因。对在肉汤培养和连续流生物膜中生长的Ng进行的QRT-PCR研究表明，LuxS的产物显著影响在生物膜形成中重要的一些基因的转录，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)、调节基因(Metr)、SIR-2家族蛋白脱乙酰基酶和MISRS。指导这一建议的假设是，群体感应和乙酰化协调调节生物膜的形成，以影响发病机制。为了验证这一假设，我们提出了以下三个具体目标：1)确定LuxS调控的基因网络中关键Ng基因的转录组以及与可逆乙酰化相关的基因。2)为了了解控制Ng生物膜发育的因素，我们将开展全面和有针对性的分析，旨在确定在生物膜和浮游状态下生长的Ng株1291的转录组、蛋白质组和乙酰组的变化；3)使用生长在HPV E6/E7转化的人宫颈上皮细胞(HCEC)上的淋球菌，我们将进行类似的转录组、蛋白质组和乙酰组的研究，以探讨细菌/真核信号在淋球菌生物膜发育中的可能作用。这些研究有可能为治疗淋病的抗生素开辟新的靶点。