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. Overall design: The starting baseline for this study is Neisseria gonorrhoeae strain NCCP11945, minimally passaged since it was genome sequenced, grown at 37C in 5% CO2. All other experiments would be based off of this, both in terms of number of samples and replicates.
Project description:Microarray comparative genome hybridization (mCGH) data was collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae, and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491, and FAM18 and N. gonorrhoeae FA1090.
Project description:Deep sequencing of cDNA from Neisseria gonorrhoeae bacteria and human neutrophils, alone and after coincubation. Overall design: Total RNA was isolated from Neisseria gonorrhoeae strains FA1090/H041 and adherent primary human neutrophils, alone and after co-culture over time. Ribosomal RNA was depleted and the remaining RNA was reverse-transcribed into a cDNA library for Illumina sequencing. Reads were mapped to the FA1090, H041 and/or human genomes.
Project description:The rise of antimicrobial-resistant Neisseria gonorrhoeae is a significant public health concern. Against this background, rapid culture-independent diagnostics may allow targeted treatment and prevent onward transmission. We have previously shown metagenomic sequencing of urine samples from men with urethral gonorrhea can recover near-complete N. gonorrhoeae genomes. However, disentangling the N. gonorrhoeae genome from metagenomic samples and robustly identifying antimicrobial resistance determinants from error-prone Nanopore sequencing is a substantial bioinformatics challenge. Here, we show an N. gonorrhoeae diagnostic workflow for analysis of metagenomic sequencing data obtained from clinical samples using R9.4.1 Nanopore sequencing. We compared results from simulated and clinical infections with data from known reference strains and Illumina sequencing of isolates cultured from the same patients. We evaluated three Nanopore variant callers and developed a random forest classifier to filter called SNPs. Clair was the most suitable variant caller after SNP filtering. A minimum depth of 20× reads was required to confidently identify resistant determinants over the entire genome. Our findings show that metagenomic Nanopore sequencing can provide reliable diagnostic information in N. gonorrhoeae infection.
Project description:Neisseria gonorrhoeae is an obligate human pathogen that is the etiological agent of gonorrhea. We explored variations in the genes of a multidrug-resistant N. gonorrhoeae isolate from a Korean patient in an effort to understand the prevalence, antibiotic resistance, and importance of horizontal gene transfer within this important, naturally competent organism. Here, we report the complete annotated genome sequence of N. gonorrhoeae strain NCCP11945.
Project description:Neisseria gonorrhoeae is the etiological agent of gonorrhea, the second most common notifiable disease in the United States. Here, we used a hybrid approach combining Oxford Nanopore Technologies MinION and Illumina MiSeq sequencing data to obtain closed genome sequences of nine clinical N. gonorrhoeae isolates.
Project description:Neisseria meningitidis and Neisseria gonorrhoeae give rise to dramatically different diseases. Their interactions with the host, however, do share common characteristics: they are both human pathogens which do not survive in the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that they have common properties that might not be found in nonpathogenic bacteria belonging to the same genetically related group, such as Neisseria lactamica. Their common properties may be determined by chromosomal regions found only in the pathogenic Neisseria species. To address this issue, we used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109-11114, 1996) to identify sequences of DNA specific for pathogenic neisseriae and not found in N. lactamica. Sequences present in N. lactamica were physically subtracted from the N. meningitidis Z2491 sequence and also from the N. gonorrhoeae FA1090 sequence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with the three species, and only those which reacted with both N. meningitidis and N. gonorrhoeae (i.e., not specific to either one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromosomes of both N. meningitidis and N. gonorrhoeae. The majority of the clones were arranged in clusters extending up to 10 kb, suggesting the presence of chromosomal regions common to N. meningitidis and N. gonorrhoeae which distinguish these pathogens from the commensal N. lactamica. The sequences surrounding these clones were determined from the N. meningitidis genome-sequencing project. Several clones corresponded to previously described factors required for colonization and survival at the port of entry, such as immunoglobulin A protease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrating that the subtractive clones are capable of pinpointing chromosomal regions shared by N. meningitidis and N. gonorrhoeae which are involved in common aspects of the host interaction of both pathogens.
Project description:An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination.
Project description:The mucosa is colonized with commensal Neisseria. Some of these niches are sites of infection for the STD pathogen Neisseria gonorrhoeae (Ngo). Given the antagonistic behavior of commensal bacteria toward their pathogenic relatives, we hypothesized that commensal Neisseria may negatively affect Ngo colonization. Here, we report that commensal species of Neisseria kill Ngo through a mechanism based on genetic competence and DNA methylation state. Specifically, commensal-triggered killing occurs when the pathogen takes up commensal DNA containing a methylation pattern that it does not recognize. Indeed, any DNA will kill Ngo if it can enter the cell, is differentially methylated, and has homology to the pathogen genome. Consistent with these findings, commensal Neisseria elongata accelerates Ngo clearance from the mouse in a DNA-uptake-dependent manner. Collectively, we propose that commensal Neisseria antagonizes Ngo infection through a DNA-mediated mechanism and that DNA is a potential microbicide against this highly drug-resistant pathogen.
Project description:The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes.