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Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni.

ABSTRACT: CmeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically important antibiotics in Campylobacter. This study aimed to identify the optimal target sites for the inhibition of CmeABC with antisense peptide nucleic acid (PNA).Eighteen PNAs were designed to bind to the translational initiation regions of cmeABC, spanning the ribosome-binding site (RBS) and the start codon of the cmeABC genes. Campylobacter jejuni was treated with CmeABC-specific PNAs (CmeABC-PNAs) at various concentrations and subjected to western blotting to measure changes in the level of CmeABC expression. The MICs of ciprofloxacin and erythromycin were measured to evaluate the impact of CmeABC knockdown on antibiotic susceptibility.While antisense PNA significantly affected CmeA and CmeB expression, interestingly, CmeC expression was not altered by any of the CmeC-PNAs used in this study. A CmeA-PNA targeting the RBS of cmeA and its upstream region reduced CmeA expression most efficiently, and CmeB expression was most significantly decreased by PNA binding to the RBS of cmeB and its downstream region. CmeA- and CmeB-PNAs increased the susceptibility of C. jejuni to ciprofloxacin and erythromycin in proportion to the inhibition levels observed in western blotting.The cmeA gene is the best target to knockdown CmeABC with antisense PNA. The RBS is the major target for the PNA-mediated antisense inhibition of CmeABC. However, regions in its vicinity also significantly influence the effectiveness of the PNA-based knockdown of CmeABC.

SUBMITTER: Oh E

PROVIDER: S-EPMC3886934 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni.

Oh Euna E Zhang Qijing Q Jeon Byeonghwa B

The Journal of antimicrobial chemotherapy 20131001 2

<h4>Objectives</h4>CmeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically important antibiotics in Campylobacter. This study aimed to identify the optimal target sites for the inhibition of CmeABC with antisense peptide nucleic acid (PNA).<h4>Methods</h4>Eighteen PNAs were designed to bind to the translational initiation regions of cmeABC, spanning the ribosome-binding site (RBS) and the start codon of the cmeABC genes. Campylobacter ...[more]

PMID: 24084637

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Project description:Campylobacter jejuni is a foodborne pathogen that is recognized as the leading cause of human bacterial gastroenteritis. The widespread use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter In addition to a role in multidrug resistance (MDR), the Campylobacter CmeABC resistance-nodulation-division (RND)-type efflux pump may be involved in virulence. As a vehicle for pathogenic microorganisms, the protozoan Acanthamoeba is a good model for investigations of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The interaction between C. jejuni 81-176 and Acanthamoeba polyphaga was investigated in this study by using a modified gentamicin protection assay. In addition, a possible role for the CmeABC MDR pump in this interaction was explored. Here we report that this MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni in A. polyphaga but is dispensable for biofilm formation and motility.IMPORTANCE The endosymbiotic relationship between amoebae and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. In this study, we found that Campylobacter jejuni was able to survive and to multiply inside Acanthamoeba polyphaga; since these microorganisms can coexist in the same environment (e.g., on poultry farms), the latter may increase the risk of infection with Campylobacter Our data suggest that, in addition to its role in antibiotic resistance, the CmeABC MDR efflux pump plays a role in bacterial survival within amoebae. Furthermore, we demonstrated synergistic effects of the CmeABC MDR efflux pump and TetO on bacterial resistance to tetracycline. Due to its role in both the antibiotic resistance and the virulence of C. jejuni, the CmeABC MDR efflux pump could be considered a good target for the development of antibacterial drugs against this pathogen.

Project description:Objectives: The aim of the study was to characterize (-)-Î±-Pinene, which is one of the chemical constituents of Alpinia katsumadai seed essential oil responsible for its resistance modulatory activity in Campylobacter jejuni. Methods: Broth microdilution method was used to evaluate the antimicrobial and resistance modulatory potential of (-)-Î±-Pinene and ethidium bromide accumulation assay to determine its efflux-inhibitory activity in subinhibitory concentration. The target efflux system was identified using knock-out mutants in several efflux related genes. Furthermore, the influence of subinhibitory concentration of (-)-Î±-Pinene on C. jejuni NCTC 11168 was investigated using microarray technology in order to elucidate the adaptive mechanism of bacteria to treatment with this phytochemical. Knock-out mutants of key adaptation genes were constructed and their role in adaptation to several stress factors, including (-)-Î±-Pinene, different osmolites and pH, was investigated using Biolog phenotypical microarrays and CFU counts. Results: (-)-Î±-Pinene was confirmed as highly efficient Campylobacter jejuni resistance modulator, due to its efflux inhibitory activity, which was significantly higher compared to reference inhibitors CCCP and reserpine. The CmeABC along with newly characterized CmeI (Cj1687) was confirmed as its main target efflux system. The transcriptional analysis indicated that the heat shock regulators HspR and HrcA are the main transcription regulators involved in adaptation to (-)-Î±-Pinene treatment. Conclusions: (-)-Î±-Pinene is a novel CmeABC and CmeI efflux inhibitor, which evokes the heat shock response in Campylobacter jejuni. Influence of (-)-Î±-Pinene on gene expression in C. jejuni NCTC 11168 was determined by expressional analysis and qRT-PCR. Exponential phase culture was adjusted to OD600=0.2 in MHB using spectrophotometer (Smart Spec, Bio-Rad, Hercules, CA, USA). Five ml of culture was treated with 62.5 mg/L of (-)-alpha-pinene dissolved in DMSO. Only DMSO (0.048 %) was added to untreated samples. Cultures were treated for 2 h, incubated microaerobically shaking (160 rpm) at 42Â°C. Experiments were carried out in 4 biological replicates. RNA Protect Bacteria reagent (Qiagen, Maryland, USA) was added to the culture and total RNA was isolated using RNeasy mini kit (Qiagen) and treated with AmbionÂ® Turbo DNA-freeTM kit (Invitrogen, USA). Microarrays with 4751 probes targeting 1756 genes specific for Campylobacter jejuni subsp. jejuni NCTC 11168 (Mycroarray, Biodiscovery-LLC, MI, USA) were used for gene expression analysis. The cDNA was synthesized with random hexamers, SuperScriptTM III Reverse Transcriptase and amynoallyl dUTP (all supplied by Invitrogen, USA) and labeled with monofunctional NHS-ester dye Amersham C3 or Cy5 (GE Healthcare, Buckinghamshire, UK). Concentration of cDNA and labeling efficiency was determined spectrophotometrically with NanoDrop 1000. Four biological replicates were hybridized to four microarrays according to manufacturerâs protocol, incubated for 24 hours at 42â°C and scanned at 532-nm (Cy3) and 635-nm (Cy5) wavelengths using GenePix 4100A (Molecular Devices, Sunnyvale, CA) following the manufacturer's protocol. Fluorescence intensities of each spot were extracted using GenePix Pro 7.0 (Molecular Devices).

Dataset Information

Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni.

Publications

Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni.

Similar Datasets

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