Project description:Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently asymptomatic in women, often leading to chronic infections. One factor contributing to this may be biofilm formation. N. gonorrhoeae can form biofilms over glass and plastic surfaces. There is also evidence that biofilm formation may occur during natural cervical infection. To further study the mechanism of this biofilm formation, transcriptional profiles of N. gonorrhoeae biofilm were compared to planktonic profiles. Biofilm RNA was extracted from N. gonorrhoeae 1291 grown for 48 hours in continuous flow chambers over glass. Planktonic RNA was extracted from the biofilm runoff. When biofilm was compared to planktonic growth, 3.8 % of the genome was differentially regulated. Genes highly up-regulated in biofilm included aniA, norB, and ccp, which play critical roles in anaerobic metabolism and oxidative stress tolerance. Down-regulated genes included the nuo gene cluster (NADH dehydrogenase) and the cytochrome bcI complex, which are involved in aerobic respiration and are thought to contribute to endogenous oxidative stress. Furthermore, we determined that aniA, ccp, and norB insertional mutants are attenuated for biofilm formation over glass and transformed human cervical epithelial cells (THCEC). This data suggests that biofilm formation could minimize oxidative stress during cervical infection and allow N. gonorrhoeae to maintain a nitric oxide steady state that may be anti-inflammatory.
Project description:Gonorrhea occurs at high incidence worldwide and has a major impact on reproductive and neonatal health worldwide. Alarmingly, with each new antibiotic introduced for gonorrhea, resistance has emerged, including resistance to penicillin, tetracycline, fluoroquinolones, and recently the third-generation cephalosporins. Treatment options are currently seriously limited and the development of a gonorrhea vaccine is a critical, longterm solution to this problem. Progress on gonorrhea vaccines has been slow, however, in part due to the high number of surface molecules in Neisseria gonorrhoeae (GC) that undergo phase or antigenic variation and a lack of understanding of protective responses. Gonorrhea vaccine development can therefore benefit from a comprehensive, unbiased approach for antigen discovery. Here we identified cell envelop proteins from Neisseria gonorrhoeae exposed to physiology relevant conditions: presence of human serum, iron limitation and anaerobic growth.
Project description:Neisseria gonorrhoeae is the causative agent of gonorrhea, a leading sexually transmitted disease with severe complications on reproductive health. The U.S. Centers for Disease Control and Prevention has categorized the public health threat induced by N. gonorrhoeae as “urgent”, due to the ease of transmission and the fast emergence of multi-drug resistant strains. The need for development of vaccines and understanding the underlying factors leading to antibiotic resistance is of utmost importance. The proteomic profiles of the 14 WHO N. gonorrhoeae reference strains have been compared to the WHO F reference strain using a mass spectrometry with tandem mass tags (TMT) labeling to analyze the cell envelope and the cytoplasmic fractions extracted from each strain. Identifying novel vaccine candidates and proteomic signatures for antimicrobial resistance will further our understanding of N. gonorrhoeae proteotypes, in relationship to their respective genotypes and phenotypes, and provide deep insights that will impact the development of preventive and therapeutic tools to combat gonorrhea.
Project description:Neisseria gonorrhoeae (GC) is a human-specific pathogen, and the agent of a sexually transmitted disease, gonorrhea. There is a critical need for new approaches to study and treat GC infections because of the growing threat of multidrug-resistant isolates and the lack of a vaccine. Despite the implied role of the GC cell envelope and membrane vesicles in colonization and infection of human tissues and cell lines, comprehensive studies have not been undertaken to elucidate their constituents. Accordingly, in pursuit of novel molecular therapeutic targets, we have applied isobaric tagging for absolute quantification coupled with liquid chromatography and mass spectrometry for proteome quantitative analyses. Mining the proteome of cell envelopes and native membrane vesicles revealed 533 and 168 common proteins, respectively, in analyzed GC strains FA1090, F62, MS11, and 1291.
Project description:The overall goals and objectives of this study are to investigate the transcriptomics of Neisseria gonorrhoeae using RNA-seq. This work will look at gene expression, start points of transcription, transcriptional termination, and differences between these in different conditions and between strains and growing cultures over time.
Project description:Transcriptional profiling of N. gonorrhoeae comparing wild type cells to cells with inactivated by chloramphenicol cassette (cm) dam replacing gene (drg) or wild type cells comparing to cells with inserted dam gene. The Goal was to study the role of drg or dam presence in overall expression profile.
Project description:Naturally occurring mtrR mutants of gonococci displaying clinically relevant levels of antibiotic resistance are often isolated from patients and mtrR mutants have been reported to be more fit than the wild type parent strain in a murine vaginal infection model. DNA-binding proteins, such as MtrR, that negatively regulate bacterial efflux pump genes have been considered to be “local” gene regulators, although there is increasing evidence that they can directly or indirectly influence expression of other genes. To define the regulatory properties of MtrR we employed microarray analysis of isogenic MtrR-positive and MtrR-negative gonococci. Keywords: single time point
Project description:Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between N. gonorrhoeae strains, but did not enhance transfer of a genetic marker.