Trans-Translation inhibitors bind to a novel site on the ribosome and clear Neisseria gonorrhoeae in vivo.
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ABSTRACT:
SUBMITTER: Aron ZD
PROVIDER: S-EPMC7979765 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
2016-05-24 | GSE73032 | GEO
Project description:BACKGROUND:While methods for annotation of genes are increasingly reliable, the exact identification of translation initiation sites remains a challenging problem. Since the N-termini of proteins often contain regulatory and targeting information, developing a robust method for start site identification is crucial. Ribosome profiling reads show distinct patterns of read length distributions around translation initiation sites. These patterns are typically lost in standard ribosome profiling analysis pipelines, when reads from footprints are adjusted to determine the specific codon being translated. RESULTS:Utilising these signatures in combination with nucleotide sequence information, we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic translatomes, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and extensions of previously annotated coding sequences. These re-annotations are supported by the presence of structural and sequence-based features next to N-terminal peptide evidence. Finally, our model identifies 61 novel genes previously undiscovered in the Salmonella enterica genome. CONCLUSIONS:Signatures within ribosome profiling read length distributions can be used in combination with nucleotide sequence information to provide accurate genome-wide identification of translation initiation sites.
| S-EPMC5576327 | biostudies-literature
Project description:Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of N. gonorrhoeae necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of N. gonorrhoeae anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC50 value of C7-3 and C7-3m2 against anaerobically grown N. gonorrhoeae was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development.
| S-EPMC5527597 | biostudies-literature
Project description:PurposeTo advance fundamental biological and translational research with the bacterium Neisseria gonorrhoeae through the prediction of novel small molecule growth inhibitors via naïve Bayesian modeling methodology.MethodsInspection and curation of data from the publicly available ChEMBL web site for small molecule growth inhibition data of the bacterium Neisseria gonorrhoeae resulted in a training set for the construction of machine learning models. A naïve Bayesian model for bacterial growth inhibition was utilized in a workflow to predict novel antibacterial agents against this bacterium of global health relevance from a commercial library of >105 drug-like small molecules. Follow-up efforts involved empirical assessment of the predictions and validation of the hits.ResultsSpecifically, two small molecules were found that exhibited promising activity profiles and represent novel chemotypes for agents against N. gonorrrhoeae.ConclusionsThis represents, to the best of our knowledge, the first machine learning approach to successfully predict novel growth inhibitors of this bacterium. To assist the chemical tool and drug discovery fields, we have made our curated training set available as part of the Supplementary Material and the Bayesian model is accessible via the web. Graphical Abstract.
| S-EPMC8274436 | biostudies-literature
Project description:Neisseria gonorrhoeae, a Gram-negative obligate human pathogenic bacterium, infects human epithelial cells and causes sexually transmitted diseases. Emerging multi-antibiotic resistant gonococci and increasing numbers of infections complicate the treatment of infected patients. Here, we used an shRNA library screen and next-generation sequencing to identify factors involved in epithelial cell infection. Folliculin (FLCN), a 64 kDa protein with a tumor repressor function was identified as a novel host factor important for N. gonorrhoeae survival after uptake. We further determined that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for its survival in the cells by modulating autophagy. In addition, FLCN was also required to maintain cell to cell contacts in the epithelial layer. In an infection model with polarized cells, FLCN inhibited the polarized localization of E-cadherin and the transcytosis of gonococci across polarized epithelial cells. In conclusion, we demonstrate here the connection between FLCN and bacterial infection and in particular the role of FLCN in the intracellular survival and transcytosis of gonococci across polarized epithelial cell layers.
| S-EPMC7499807 | biostudies-literature
Project description:Tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the 'tmRNA-SmpB' system (transfer messenger RNA-small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon-anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon-anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for DeltasmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.
| S-EPMC2637316 | biostudies-literature
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.
2017-02-09 | GSE60347 | GEO
Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in widespread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods.
| S-EPMC4176156 | biostudies-literature
Project description:The bacterial pathogen Neisseria gonorrhoeae (Gc) infects mucosal sites rich in antimicrobial proteins, including the bacterial cell wall-degrading enzyme lysozyme. Certain Gram-negative bacteria produce protein inhibitors that bind to and inhibit lysozyme. Here, we identify Ng_1063 as a new inhibitor of lysozyme in Gc, and we define its functions in light of a second, recently identified lysozyme inhibitor, Ng_1981. In silico analyses indicated that Ng_1063 bears sequence and structural homology to MliC-type inhibitors of lysozyme. Recombinant Ng_1063 inhibited lysozyme-mediated killing of a susceptible mutant of Gc and the lysozyme-sensitive bacterium Micrococcus luteus. This inhibitory activity was dependent on serine 83 and lysine 103 of Ng_1063, which are predicted to interact with lysozyme's active site residues. Lysozyme co-immunoprecipitated with Ng_1063 and Ng_1981 from intact Gc. Ng_1063 and Ng_1981 protein levels were also increased in Gc exposed to lysozyme. Gc lacking both ng1063 and ng1981 was significantly more sensitive to killing by lysozyme than wild-type or single mutant bacteria. When exposed to human tears or saliva, in which lysozyme is abundant, survival of Δ1981Δ1063 Gc was significantly reduced compared to wild-type, and survival was restored upon addition of recombinant Ng_1981. Δ1981Δ1063 mutant Gc survival was additionally reduced in the presence of human neutrophils, which produce lysozyme. We found that while Ng_1063 was exposed on the surface of Gc, Ng_1981 was both in an intracellular pool and extracellularly released from the bacteria, suggesting that Gc employs these two proteins at multiple spatial barriers to fully neutralize lysozyme activity. Together, these findings identify Ng_1063 and Ng_1981 as critical components for Gc defense against lysozyme. These proteins may be attractive targets for antimicrobial therapy aimed to render Gc susceptible to host defenses and/or for vaccine development, both of which are urgently needed against drug-resistant gonorrhea.
| S-EPMC6033460 | biostudies-literature
Project description:BackgroundNeisseria gonorrhoeae (GC) is a Gram-negative pathogen that most commonly infects mucosal surfaces, causing sexually transmitted urethritis in men and endocervicitis in women. Serious complications associated with these infections are frequent and include pelvic inflammatory disease, ectopic pregnancy, and infertility. The incidence of gonorrhea cases remains high globally while antibiotic treatment options, the sole counter measures against gonorrhea, are declining due to the remarkable ability of GC to acquire resistance. Evaluating of potential drug targets is essential to provide opportunities for developing antimicrobials with new mechanisms of action. We propose the GC Obg protein, belonging to the Obg/CgtA GTPase subfamily, as a potential target for the development of therapeutic interventions against gonorrhea, and in this study perform its initial functional and biochemical characterization.ResultsWe report that NGO1990 encodes Obg protein, which is an essential factor for GC viability, associates predominantly with the large 50S ribosomal subunit, and is stably expressed under conditions relevant to infection of the human host. The anti-Obg antisera cross-reacts with a panel of contemporary GC clinical isolates, demonstrating the ubiquitous nature of Obg. The cellular levels of Obg reach a maximum in the early logarithmic phase and remain constant throughout bacterial growth. The in vitro binding and hydrolysis of the fluorescent guanine nucleotide analogs mant-GTP and mant-GDP by recombinant wild type and T192AT193A mutated variants of Obg are also assessed.ConclusionsCharacterization of the GC Obg at the molecular and functional levels presented herein may facilitate the future targeting of this protein with small molecule inhibitors and the evaluation of identified lead compounds for bactericidal activity against GC and other drug-resistant bacteria.
| S-EPMC4487204 | biostudies-literature