Global RNA profiles show target selectivity and physiological effects of peptide-delivered antisense antibiotics
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ABSTRACT:
ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium
PROVIDER: GSE155764 | GEO | 2021/03/19
REPOSITORIES: GEO
Similar Datasets
Project description:Antisense peptide nucleic acids (PNAs) that target mRNAs of essential bacterial genes exhibit specific bactericidal effects in several microbial species, but our mechanistic understanding of PNA activity and their target gene spectrum is limited. Here, we present a systematic analysis of PNAs targeting eleven essential genes with varying expression levels in uropathogenic Escherichia coli (UPEC). We demonstrate that UPEC is susceptible to killing by peptide-conjugated PNAs, especially when targeting the widely-used essential gene acpP. Our evaluation yields three additional promising target mRNAs for effective growth inhibition, i.e., dnaB, ftsZ, and rpsH. The analysis also shows that transcript abundance does not predict target vulnerability and that PNA-mediated growth inhibition is not universally associated with target mRNA depletion. Global transcriptomic analyses further reveal PNA sequence-dependent but also -independent responses, including the induction of envelope stress response pathways. Importantly, we show that the growth inhibitory capacity of 9mer PNAs is generally as effective as their 10mer counterparts. Overall, our systematic comparison of a range of PNAs targeting mRNAs of different essential genes in UPEC suggests important features for PNA design, reveals a general bacterial response to PNA conjugates and establishes the feasibility of using PNA antibacterials to combat UPEC.
2022-04-28 | GSE191313 | GEO
Project description:Antisense oligomers (ASOs) such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (Make AntiSense Oligomers Now), a webserver for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASON’s off-target predictions on experiments in which we treated Salmonella enterica serovar Typhimurium with a series of 10mer PNAs derived from a PNA targeting the essential gene acpP but carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to target acpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq dataset from uropathogenic Escherichia coli (UPEC) treated with eleven different PNAs confirmed our findings are not unique to Salmonella. We believe that MASON’s off-target assessment will improve the design of specific PNAs and other ASOs.
2022-07-01 | GSE199542 | GEO
Project description:Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes bear great potential in the fight against antimicrobial resistance and for precision microbiome editing. The development of such antisense antibiotics has primarily focused on using phosphoramidite morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based therapy in general. Here, we compare seven chemically distinct 10mer-ASOs, all designed to target the same essential acpP mRNA upon delivery with a KFF-peptide carrier into Salmonella, comparing PNA, PMO, phosphorothioate modified-DNA (PTO), 2’-methoxylated RNA (RNA-OMe), 2’-methoxyethylated RNA (RNA-MOE), 2’-fluorinated RNA (RNA-F) and 2’-locked RNA (LNA). Our systematic analysis using a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and bacterial killing suggests that only PNA and PMO make it into Salmonella to cause bacterial growth inhibition. However, given their high target binding and translational repression activity in vitro, ASOs based on LNA and RNA-MOE appear to be promising candidates for antisense antibiotics that will require the identification of an efficient carrier.
2023-07-11 | GSE232819 | GEO
Project description:Objectives Pyrrolobenzodiazepine (PBD) dimers, tethered through inert propyldioxy or pentyldioxy linkers, possess potent bactericidal activity against a range of Gram-positive bacteria by virtue of their capacity to cross-link duplex DNA in sequence-selective fashion. Here we attempt to improve the antibacterial activity and cytotoxicity profile of PBD-containing conjugates by extension of dimer linkers and replacement of one PBD unit with phenyl-substituted or benzo-fused heterocycles that facilitate non-covalent interactions with duplex DNA. Methods DNase I footprinting was used to identify high-affinity DNA binding sites. A staphylococcal gene microarray was used to assess epidemic methicillin-resistant Staphylococcus aureus 16 phenotypes induced by PBD conjugates. Molecular dynamics simulations were employed to investigate the accommodation of compounds within the DNA helix. Results Increasing the length of the linker in PBD dimers led to a progressive reduction in antibacterial activity, but not in their cytotoxic capacity. Complex patterns of DNA binding were noted for extended PBD dimers. Modelling of DNA strand cross-linking by PBD dimers indicated distortion of the helix. A majority (26 of 43) of PBD-biaryl conjugates possessed potent antibacterial activity with little or no helical distortion and a more favourable cytotoxicity profile. Bactericidal activity of PBD-biaryl conjugates was determined by inability to excise covalently bound drug molecules from bacterial duplex DNA. Conclusions PBD-biaryl conjugates have a superior antibacterial profile compared with PBD dimers such as ELB-21. We have identified six PBD-biaryl conjugates as potential drug development candidates. [Data is also available from http://bugs.sgul.ac.uk/E-BUGS-118]
2012-04-17 | E-BUGS-118 | biostudies-arrayexpress
Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. ChiP-Seq: M. tuberculosis H37Rv (GC1237) cultures grown in vitro to exponential phase. Two wild type samples plus one isogenic phoP mutant as control
2014-06-02 | E-GEOD-54239 | biostudies-arrayexpress
Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. RNA-Seq: M. tuberculosis H37Rv (GC1237) cultures grown in vitro to exponential phase. One wild type sample plus one isogenic phoP mutant sample
2014-06-02 | E-GEOD-54240 | biostudies-arrayexpress
Project description:To investigate the molecular mechanisms of miRNA-mRNA expression underlying this phenotype, ovarian samples from PNA and control mice were subjected to microRNA-seq and RNA-seq. Differential expression analyses were carried out to identify differentially expressed microRNAs (DEmiRs) and differentially expressed genes (DEGs). Target genes of DEmiRs were predicted by miRTarbase and visualization by Cytoscape. Functional annotation and pathway enrichment analyses for the DEGs and target genes were performed through the DAVID database. Then, to reveal the interactions involved in the pathogenesis of PCOS, the integrated analysis of miRNA-mRNA was performed in PNA mice vs. controls and granulosa cells (GCs) of PCOS women vs. health women. Protein–protein interaction (PPI) networks were established for these negatively regulated pairs via the STRING database. The expression and correlation of potential miRNAs and targets were further validated using RT-qPCR in more PNA mice and clinical human granulosa cells.
2021-03-04 | GSE168166 | GEO
Project description:Purpose: Current methods to repair ablation-type peripheral nerve injuries (PNIs) using peripheral nerve allografts (PNAs) often result in poor functional recovery due to immunological rejection as well as slow and inaccurate outgrowth of regenerating axon sprouts. In contrast, ablation-type PNIs repaired by PNAs using a multistep protocol, in which one step uses the membrane fusogen polyethylene glycol (PEG), permanently restore sciatic-mediated behaviors within weeks. Axons and cells within the PNA remain viable, even though outbred host and donor tissues are neither immunosuppressed nor tissue matched. PEG-fused PNAs exhibit significantly reduced T cell and macrophage infiltration, apoptosis, and expression of major histocompatibility complex I/II. In this study, we analyzed the coding transcriptome of PEG-fused PNAs to determine possible mechanisms underlying immunosuppression. Methods: Ablation-type sciatic PNIs in adult Sprague Dawley rats were repaired using PNAs and a PEG-fusion protocol combined with neurorrhaphy. Electrophysiological and behavioral recovery tests confirmed successful PEG-fusion of PNAs. RNA sequencing analyzed differential expression profiles of protein-coding genes between PEG-fused PNAs and NC PNAs (not treated with PEG) at 14d PO, along with Unoperated nerves. Sequencing results were validated by Quantitative Reverse Transcription PCR (RT-qPCR). Results: PEG-fused PNAs display significant downregulation of a great many gene transcripts associated with innate and adaptive allorejection responses. Many Schwann cell-associated transcripts are upregulated, and many cellular processes such as extracellular matrix remodeling, cell and tissue development are particularly enriched. Transcripts encoding several potentially immunosuppressive proteins (e.g. Thrombospondins 1 and 2) are upregulated in PEG-fused PNAs. Conclusions: This study is the first to characterize the coding transcriptome of PEG-fused PNAs, and identifies possible links between alterations of the extracellular matrix and suppression of the allorejection response. The results establish a molecular-basis to understand mechanisms underlying PEG-mediated immunosuppression.
2020-02-19 | GSE145504 | GEO
Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.
2014-06-02 | GSE54240 | GEO
Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.
2014-06-02 | GSE54239 | GEO