Neisseria gonorrhoeae is the causative agent of gonorrhea, one of the most prevalent infectious diseases in this country. In addition to local infection, manifested as urethritis or cervicitis, the gonococcus can cause serious complications, including pelvic inflammatory disease (PID), the leading cause of sterility in women, and disseminated gonococcal infection (DGI), a leading cause of septic arthritis in the U.S. There are ~350,000 cases of gonorrhea reported to the CDC each year and an estimated 65 million cases worldwide. This organism has acquired resistance to antibiotics, such that the only CDC recommended treatment for gonorrhea is third generation cephalosporins. Thus, we need alternative approaches to controlling gonorrhea, such as vaccines or topical microbicides. These alternatives require that we have a better understanding of how gonococci avoid host defense systems. We have determined that N. gonorrhoeae displays remarkable resistance to killing by reactive nitrogen species (RNS), especially peroxynitrite, and we propose that this characteristic allows this organism to escape host defense mechanisms. We also propose that N. gonorrhoeae have novel mechanisms for this resistance to peroxynitrite and that these mechanisms may be redundant.
Our Specific Aims are:
Aim 1. To identify gonococcal genes involved in resistance to peroxynitrite by cloning into E. coli. We will clone gonococcal peroxynitrite resistance genes into E. coli, identify the genes and characterize their regulation and the effects of insertional inactivation on RNS resistance.
Aim 2. Identification of the function of novel gonococcal peroxynitrite resistance gene(s). It may not be possible to clone the peroxynitrite genes in E. coli. Therefore, we will identify and purify gonococcal proteins that confer peroxynitrite resistance in vitro, identify them by MALDI-TOF, and construct mutants to determine their role in peroxynitrite resistance in vivo. We will determine the patterns of nitrotyrosine formation upon peroxynitrite exposure, the ability to repair nitrotyrosine damage as a function of time, the ability to prevent peroxynitrite-induced oxidation of dihydrorhodamine and DNA, and the ability to remove nitro- groups from nitrated bovine serum albumin. Successful completion of this project will identify novel genes involved in resistance to peroxynitrite in the gonococcus, an obligate human mucosal pathogen. This would be the first mucosal bacterium to be extensively investigated for mechanisms of peroxynitrite resistance. Thus, in addition to extending our knowledge of how gonococci avoid host defenses, we will contribute to increasing the general understanding of the role of RNS resistance in bacterial evasion of host innate immune defenses.
Neisseria gonorrhoeae is the causative agent of gonorrhea, one of the most prevalent infectious diseases in this country. In addition to local infection, manifested as urethritis or cervicitis, the gonococcus can cause serious complications, including sterility or ectopic pregnancy in women in women and arthritis in men and women. There are ~350,000 cases of gonorrhea reported to the CDC each year and an estimated 65 million cases worldwide. This organism has acquired resistance to antibiotics, such that the only CDC recommended treatment for gonorrhea is cephalosporins. We need alternative approaches to controlling gonorrhea, such as vaccines or topical microbicides. These alternatives require that we have a better understanding of how gonococci avoid host defense systems. Successful completion of this project will identify novel genes involved in resistance to peroxynitrite killing in the gonococcus, an obligate human pathogen. This would be the first mucosal bacterium to be extensively investigated for mechanisms of peroxynitrite resistance.
| Spence, Stephen A; Clark, Virginia L; Isabella, Vincent M (2012) The role of catalase in gonococcal resistance to peroxynitrite. Microbiology 158:560-70 |
| Isabella, Vincent M; Clark, Virginia L (2011) Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae. BMC Genomics 12:51 |
| Isabella, Vincent M; Clark, Virginia L (2011) Identification of a conserved protein involved in anaerobic unsaturated fatty acid synthesis in Neiserria gonorrhoeae: implications for facultative and obligate anaerobes that lack FabA. Mol Microbiol 82:489-501 |
