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Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli.

ABSTRACT: 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 11 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 9mer PNAs are generally as effective in inhibiting bacterial growth 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.

SUBMITTER: Popella L

PROVIDER: S-EPMC9226493 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli

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

Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli

Project description:Comprehensive analysis of PNA-based antisense antibiotics targeting various essential genes in uropathogenic Escherichia coli

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Synergistic Effect of Propolis and Antibiotics on Uropathogenic Escherichia coli.

Project description:Urinary tract infections (UTIs) are the most common bacterial infections around the world. Uropathogenic Escherichia coli (UPEC) is among the main pathogens isolated in UTIs. The rate of UPEC with high resistance towards antibiotics and multidrug-resistant bacteria have increased dramatically and conduct to the difficulty to treat UTIs. Due to the rarefaction of new antibiotics molecules, new alternative strategies must be evaluated. Since many years, propolis has demonstrated an interesting antibacterial activity against E. coli. Here, we evaluated its activity added to antibiotics on a panel of UPEC with different resistance mechanisms. Minimal inhibitory concentrations (MICs) and time-kill curves of fosfomycin, ceftriaxone, ertapenem and ofloxacin, with and without propolis, were determined. Significant diminution of the MICs was observed using ceftriaxone or ofloxacin + propolis. Propolis alone had a bacteriostatic activity with time-dependent effect against UPEC. The addition of this nutraceutical improved the effect of all the antibiotics evaluated (except fosfomycin) and showed a synergistic bactericidal effect (fractional inhibitory concentrations index ≤ 0.5 and a decrease ≥ 2 log CFU/mL for the combination of propolis plus antibiotics compared with the antibiotic alone). Propolis is able to restore in vitro antibiotic susceptibility when added to antibiotics against UPEC. This study showed that propolis could enhance the efficiency of antibiotics used in UTIs and could represent an alternative solution.

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Genomic Sequences of Uropathogenic Escherichia coli Strains with Various Fluoroquinolone Resistance Profiles.

Project description:The emergence of drug-resistant uropathogenic Escherichia coli (UPEC) has hampered antibiotic therapy for urinary tract infections. To elucidate the resistance mechanisms of UPEC, we performed whole-genome sequencing of eight UPEC strains with different fluoroquinolone resistance levels. Here, we report our sequencing data, providing a valuable resource for understanding such mechanisms.

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Phenotypic heterogeneity enables uropathogenic Escherichia coli to evade killing by antibiotics and serum complement.

Project description:Uropathogenic strains of Escherichia coli (UPEC) are the major cause of bacteremic urinary tract infections. Survival in the bloodstream is associated with different mechanisms that help to resist serum complement-mediated killing. While the phenotypic heterogeneity of bacteria has been shown to influence antibiotic tolerance, the possibility that it makes cells refractory to killing by the immune system has not been experimentally tested. In the present study we sought to determine whether the heterogeneity of bacterial cultures is relevant to bacterial targeting by the serum complement system. We monitored cell divisions in the UPEC strain CFT073 with fluorescent reporter protein. Stationary-phase cells were incubated in active or heat-inactivated human serum in the presence or absence of different antibiotics (ampicillin, norfloxacin, and amikacin), and cell division and complement protein C3 binding were measured by flow cytometry and immunofluorescence microscopy. Heterogeneity in the doubling times of CFT073 cells in serum enabled three phenotypically different subpopulations to be distinguished, all of them being recognized by the C3 component of the complement system. The population of rapidly growing cells resists serum complement-mediated lysis. The dominant subpopulation of cells with intermediate growth rate is susceptible to serum. The third population, which does not resume growth upon dilution from stationary phase, is simultaneously protected from serum complement and antibiotics.

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Disruption of Membrane by Colistin Kills Uropathogenic Escherichia coli Persisters and Enhances Killing of Other Antibiotics.

Project description:Persisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active against Escherichia coli persisters at high concentrations (25 or 50 μg/ml). At a clinically relevant lower concentration (10 μg/ml), colistin alone had no apparent effect on E. coli persisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugs in vitro The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.

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Uropathogenic Escherichia coli biofilm proteomics

Project description:We previously determined that loss of respiratory quinol oxidase cytochrome bd disrupts biofilm formation in uropathogenic Escherichia coli (UPEC). In this study, we extracted and interrogated the outer membrane and extracellular matrix of colony biofilms formed by UPEC isolate UTI89 and an isogenic mutant lacking cytochrome bd (∆cydAB).

2021-04-01 | PXD023998 | Pride

Targeting of the Essential acpP, ftsZ, and rne Genes in Carbapenem-Resistant Acinetobacter baumannii by Antisense PNA Precision Antibacterials.

Project description:Infections by carbapenem-resistant A. baumannii (CRAB), a widespread nosocomial pathogen, are becoming increasingly difficult to prevent and treat. Therefore, there is an urgent need for discovery of novel antibiotics against CRAB. Programmable, precision antisense antibiotics, e.g., based on the nucleic acid mimic PNA (peptide nucleic acid) have shown promise in this respect in the form of PNA-BPP (bacteria penetrating peptide) conjugates targeting essential bacterial genes. In the present study, we designed and synthesized a series of PNA-BPPs targeting the translation initiation region of the ftsZ, acpP, or rne gene of CRAB strains. The antimicrobial activity of the compounds and effects on gene expression level was compared to that of analogous mismatch PNA controls. Three antisense conjugates (KFF)3K-eg1-(acpP)PNA (5639), (KFF)3K-eg1-(ftsZ)PNA (5612), and (KFF)3-K-eg1-(rne)PNA (5656) exhibited complete growth inhibition against several CRAB strains at 1-2, 2-8, and 2 µM, respectively, and the compounds were bactericidal at 1-2× MIC. The bactericidal effect was correlated to reduction of target gene mRNA level using RT-qPCR, and the compounds showed no bacterial membrane disruption activity at 1-2× MIC. PNA5612 was tested against a series of 12 CRAB isolates and all were sensitive at 2-8 µM. In addition, the conjugates exhibited no cellular toxicity in the HepG2 cell line (up to 20 μM) and did not shown significant antibacterial activity against other Gram negatives (E. coli, P. aeruginosa). These results provide a starting point for discovery of antisense precision designer antibiotics for specific treatment of CRAB infections.

| S-EPMC8071358 | biostudies-literature

Effects of LPS Composition in <i>Escherichia coli</i> on Antibacterial Activity and Bacterial Uptake of Antisense Peptide-PNA Conjugates.

Project description:The physical and chemical properties of the outer membrane of Gram-negative bacteria including Escherichia coli have a significant impact on the antibacterial activity and uptake of antibiotics, including antimicrobial peptides and antisense peptide-peptide nucleic acid (PNA) conjugates. Using a defined subset of E. coli lipopolysaccharide (LPS) and envelope mutants, components of the LPS-core, which provide differential susceptibility toward a panel of bacterial penetrating peptide (BPP)-PNA conjugates, were identified. Deleting the outer core of the LPS and perturbing the inner core only sensitized the bacteria toward (KFF)3K-PNA conjugates, but not toward conjugates carrying arginine-based BPPs. Interestingly, the chemical composition of the outer LPS core as such, rather than overall hydrophobicity or surface charge, appears to determine the susceptibility to different BPP-PNA conjugates thereby clearly demonstrating the complexity and specificity of the interaction with the LPS/outer membrane. Notably, mutants with outer membrane changes conferring polymyxin resistance did not show resistance toward the BPP-PNA conjugates, thereby eliminating one possible route of resistance for these molecules. Finally, envelope weakening, through deletion of membrane proteins such as OmpA as well as some proteins previously identified as involved in cationic antimicrobial peptide uptake, did not significantly influence BPP-PNA conjugate activity.

| S-EPMC9251361 | biostudies-literature

Activating the Cpx response induces tolerance to antisense PNA delivered by an arginine-rich peptide in <i>Escherichia coli</i>.

Project description:Cell-penetrating peptides (CPPs) are increasingly used for cellular drug delivery in both pro- and eukaryotic cells, and oligoarginines have attracted special attention. How arginine-rich CPPs translocate across the cell envelope, particularly for prokaryotes, is still unknown. Arginine-rich CPPs efficiently deliver antimicrobial peptide nucleic acid (PNA) to its intracellular mRNA target in bacteria. We show that resistance to PNA conjugated to an arginine-rich CPP in Escherichia coli requires multiple genetic modifications and is specific for the CPP part and not to the PNA part. An integral part of the resistance was the constitutively activated Cpx-envelope stress response system (cpx∗), which decreased the cytoplasmic membrane potential. This indicates an indirect energy-dependent uptake mechanism for antimicrobials conjugated to arginine-rich CPPs. In agreement, cpx∗ mutants showed low-level resistance to aminoglycosides and an arginine-rich CPP conjugated to a peptide targeting the DNA sliding clamp, i.e., similar uptake in E. coli for these antimicrobial compounds.

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