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.
Recent reports of gonococci resistant to ceftriaxone and azithromycin have led the WHO to declare a health emergency. Recently, CDC recommended that oral therapy for gonorrhea be discontinued in the USA. It is recognized that new drugs are needed for treatment of gonorrhea. The studies in this grant form the basis for targeting deacetylase and acetyltransferase as novel therapy in treatment of gonorrhea.
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