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ABSTRACT:
PROVIDER: PRJEB46053 | ENA |
REPOSITORIES: ENA
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Project description:The Proline-rich Antimicrobial Peptide (PrAMP) apidaecin (Api) inhibits translation by binding in the ribosomal nascent peptide exit tunnel, trapping release factors RF1 or RF2, and arresting ribosomes at stop codons. To explore the extent of sequence variations of the native 18-amino acid Api that allows it to preserve its activity, we screened a library of synthetic mutant Api genes expressed in bacterial cells, resulting in nearly 350,000 peptide variants with multiple substitutions. By applying orthogonal negative and positive selection strategies, we identified a number of multi-substituted Api variants capable of arresting ribosomes at stop codons. Our findings underscore the critical contribution of specific amino acid residues of the peptide for its on-target function while significantly expanding the variety of PrAMPs acting on the terminating ribosome. Additionally, some of the tested synthesized multi-substituted Api variants exhibit improved antibacterial activity compared to that of the wild type PrAMP and may constitute the starting point to develop clinically useful antimicrobials.
2024-06-18 | GSE269894 | GEO
Project description:In search for peptides with higher or special binding affinity and for further understanding of the mode of action, a full substitutional analysis of peptide PeB using microarrays was performed. Thus, 152 PeB mutant variants were generated. In each of them, the full-length sequence was preserved except for only one amino acid from the eight loop-forming amino acids of the original PeB peptide (ARDFYDYDVFYYAMD) which was substituted with the 19 remaining natural amino acids. To assess binding, influenza material was labeled with a protein reacting fluorophore.
2016-02-27 | GSE78700 | GEO
Project description:Pseudomonas species are ubiquitous in plant-associated environments and produce an array of volatiles, enzymes and antimicrobials. The biosynthesis of many metabolites is regulated by the GacS/GacA two-component regulatory system. Transcriptome analysis of Pseudomonas fluorescens SBW25 revealed that 702 genes were differentially regulated (fold change>4, P<0.0001) in a gacS::Tn5 mutant, with 300 and 402 genes up- and down-regulated, respectively. Genes that were significantly down-regulated are involved in viscosin biosynthesis (viscABC), protease production (aprA), motility, biofilm formation, and secretory systems. Genes that were significantly up-regulated are involved in siderophore biosynthesis and oxidative stress. In contrast to previous studies with gac-mutants of other Pseudomonas species/strains, the gacS mutant of SBW25 inhibited growth of oomycete, fungal and bacterial pathogens significantly more than parental strain SBW25. A potential candidate for this enhanced antimicrobial activity was a large nonribosomal peptide synthetase (NRPS) gene cluster predicted to encode for an 8-amino-acid ornicorrugatin-like peptide. Site-directed mutagenesis of an NRPS gene in this cluster, however, did not lead to a reduction in the antimicrobial activity of the gacS mutant. Collectively these results indicate that a mutation in the GacS/GacA regulatory system causes major transcriptional changes in P. fluorescens SBW25 and significantly enhances its antimicrobial activities by yet unknown mechanisms. This expression study used total RNA recovered from four separate wild-type cultures of Pseudomonas fluorescens SBW25 and four separate cultures of the gacS mutant. Expression design was based on the updated genome sequence of Pseudomonas fluorescens SBW25, NC_012660.1 and associated plasmid pQBR0476 with nineteen 60-mer probe per gene. Each probe is replicated 3 times. The design includes random GC and other control probes.
2013-06-17 | E-GEOD-43443 | biostudies-arrayexpress
Project description:A short sequence of 11 amino acids belonging to the cj0669 protein from Campylobacter jejuni NCTC 11168, which was previously identified as potentially immunogenic, was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence. Twelve peptides, one representing the original sequence and eleven peptides with each residue replaced by alanine in turn, were synthesized on microarrays by JPTs Pepstar Technology. For each microarray, nine replicates for each peptide were spotted. The microarray was separated into three incubation chambers by the ProPlate 3-well module (Grace Biolabs) to allow for incubation with different antibodies in parallel. For specific interactions, rabbit polyclonal IgG to C. jejuni was used, while unspecific binding to the epitope sequence was checked using rabbit polyclonal IgG to Salmonella enterica.
2013-04-06 | E-GEOD-45556 | biostudies-arrayexpress
Project description:A short sequence of 11 amino acids belonging to the cj0669 protein from Campylobacter jejuni NCTC 11168 which was previously identified as potentially immunogenig was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence. Twelve peptides, one representing the original sequence and eleven peptides with each residue replaced by alanine in turn, were synthesized on microarrays by JPTs Pepstar Technology. For each microarray, nine replicates for each peptide were spotted. The microarray was seperated into three incubation chambers by the ProPlate 3-well module (Grace Biolabs) to allow for incubation with different antibodies in parallel. For specific interaction rabbit polyclonal IgG to C.jejuni was used, while non-specific binding to the epitope sequence was checked using rabbit polyclonal IgG to H. pylori (Abcam ab20459).
2013-05-06 | E-GEOD-46651 | biostudies-arrayexpress
Project description:Ribosome translates mRNAs by matching every 3-nucleotide sequence in mRNA, producing the appropriate proteins. As the amino acid sequence directly dictates the activity of the protein, frameshift often leads to unwanted effects. Here, we demonstrate that the intracellular pathogen Salmonella Typhimurium suppresses frameshift in the ugtL antimicrobial resistance gene during translation. This frameshift suppression is mediated by a ribosome pause occurring in a newly-identified overlapping gene, serving as a non-slip bump. Given that the pause site contains a poly-proline motif and can be resolved by elongation factor P, the removal of the ribosome pause by substituting the motif induces ribosome slippage in ugtL, resulting in nonfunctional UgtL frameshifted protein production. This renders Salmonella sensitive to antimicrobial peptides, indicating that elongation factor P-dependent ribosome pause is required for full antimicrobial resistance. These findings reveal a new regulatory mechanism of ribosome pause to ensure functional protein production by suppressing ribosome slippage-mediated frameshift.
2026-08-17 | GSE277383 | GEO
Project description:Ribosome translates mRNAs by matching every 3-nucleotide sequence in mRNA, producing the appropriate proteins. As the amino acid sequence directly dictates the activity of the protein, frameshift often leads to unwanted effects. Here, we demonstrate that the intracellular pathogen Salmonella Typhimurium suppresses frameshift in the ugtL antimicrobial resistance gene during translation. This frameshift suppression is mediated by a ribosome pause occurring in a newly-identified overlapping gene, serving as a non-slip bump. Given that the pause site contains a poly-proline motif and can be resolved by elongation factor P, the removal of the ribosome pause by substituting the motif induces ribosome slippage in ugtL, resulting in nonfunctional UgtL frameshifted protein production. This renders Salmonella sensitive to antimicrobial peptides, indicating that elongation factor P-dependent ribosome pause is required for full antimicrobial resistance. These findings reveal a new regulatory mechanism of ribosome pause to ensure functional protein production by suppressing ribosome slippage-mediated frameshift.
2026-08-17 | GSE277235 | GEO
Project description:In search for peptides with higher or special binding affinity and for further understanding of the mode of action, a full substitutional analysis of peptide PeB using microarrays was performed. Thus, 152 PeB mutant variants were generated. In each of them, the full-length sequence was preserved except for only one amino acid from the eight loop-forming amino acids of the original PeB peptide (ARDFYDYDVFYYAMD) which was substituted with the 19 remaining natural amino acids. To assess binding, influenza material was labeled with a protein reacting fluorophore. Microarray-based substitutional analysis of peptide PeB was performed using a PepStar® peptide library spotted on glass slides by JPT Peptide Technologies. The slides were used without additional treatment. For the labeling of proteins with a fluorescent dye, Dyomics DY-634 (λex = 635 nm, λem = 654 nm, Fluoro-spin 634 Kit (emp Biotech) was used, according to the manufacturer´s instructions. The following materials were labeled: NewYork H3N2, Aichi H3N2, Victoria H3N2 and California H1N1. Labeled analytes were incubated several hours or overnight at indicated concentrations using Femtotip buffer (FTP)30 (20 mM Tris, 30% glycerol, 3% polyvinylpyrrolidon 90, 0.1% Tween 20, pH 8.4) for dilution. The slides were washed twice in FTP and twice in ultrapure water and subsequently dried under a stream of nitrogen. Experiments were performed in triplicates using glycans (2,3'-/2,6'-sialyllactose) and proteins (Anti-H1/Anti-H3 antibodies, fetuin) as positive and negative controls.
2016-02-27 | E-GEOD-78700 | biostudies-arrayexpress