A global RNA analysis of Neisseria gonorrhoeae in vitro and during human infection

Thesis (Ph.D.)--Boston UniversityThe mucosal disease, gonorrhea, caused by the Gram-negative pathogen Neisseria gonorrhoeae, is estimated to have at least 700,000 cases annually in the United States and 62 million cases worldwide. A strict human pathogen, N. gonorrhoeae infects several mucosal sites throughout the body making proper gene regulation crucial. The goal of these studies was to define the global transcriptional response of N. gonorrhoeae during infection by analyzing its transcriptome during in vitro growth, during incubation with human epithelial cells, and during in vivo mucosal infection of the human female genital tract. Using RNA sequencing, we identified several new small RNA transcripts expressed in vitro that have the potential to regulate target mRNAs. Our studies were aided by the development of a novel computer program, Rockhopper, designed specifically for analysis of prokaryotic transcriptomes. Secondary methods were used to corroborate a strong correlation between Rockhopper analysis and N. gonorrhoeae transcriptional start sites, operon structures and gene expression levels. We also utilized Rockhopper to analyze the gonococcal transcriptome expressed during incubation with a human endocervical cell line. During such incubation, N. gonorrhoeae was demonstrated to regulate a large number of stress response and respiratory genes. Corresponding analysis of host cells during incubation with N. gonorrhoeae revealed increased expression of host pathways involved in innate immunity, adaptive immunity, cancer and apoptosis. Finally, analysis of gonococcal RNA from four vaginal lavage samples of female patients exposed to partners infected with N. gonorrhoeae was performed. This analysis demonstrated a similar profile of gonococcal stress response genes compared to incubation with epithelial cells. In addition, several novel sRNAs expressed by the gonococcus only during in vivo infection were also identified. Analysis of the same vaginal lavage samples demonstrated that a number of human genes involved in immune pathways and cancer are expressed during mucosal gonococcal infection. These studies are the first to analyze gene regulation in N. gonorrhoeae globally during infection and greatly expand our knowledge of how the host and pathogen respond to infection. Furthermore, they have the potential to aid in the development of novel antibacterial therapeutics or new vaccine targets for this disease