Lysine Acetylation in N. gonorrhoeae Quorum Sensing and Biofilm Formation

描述（由申请者提供）：Apicella、Gibson、Kirby和Wolfe实验室进行了独特的观察，发现淋病奈瑟菌蛋白经历了广泛的Nϵ-lysine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。利用新的质谱分析技术，吉布森博士的实验室已经证明，luxS操纵子和ackA的突变体表现出乙酰化蛋白的显著增加。其中一种乙酰化的蛋白是反应调节因子MisR，它与其同源的传感器激酶MisS合作形成双组分信号转导系统（TCS）。我们的数据强烈表明，MisRS能感知LuxS的产品。我们的初步结果表明，luxS操纵子通过MisRS作为一个经典的群体感应系统起作用，并且该TCS控制调节细胞分裂和Nϵ-lysine乙酰化的基因。在肉汤培养和连续流生物膜中培养Ng的qRT-PCR研究表明，luxS的产物显著影响了许多在生物膜形成中重要基因的转录，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）、调节基因（metR）、SIR-2家族蛋白去乙酰化酶和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，影响发病机制。为了验证这一假设，我们提出了以下3个具体目标：1)确定luxs调控基因网络中关键Ng基因的转录组以及可逆乙酰化相关基因。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，旨在确定在生物膜和浮游条件下生长的Ng菌株1291的转录组，蛋白质组和乙酰化组的变化。3)使用在HPV E6/E7转化的人宫颈上皮细胞（HCEC）上生长的淋病奈球菌。我们将对转录组、蛋白质组和乙酰组进行类似的研究，以探究细菌/真核信号在淋病奈瑟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一系列新的靶点。

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.