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Antimicrobial action of the cyclic peptide bactenecin on Burkholderia pseudomallei correlates with efficient membrane permeabilization.

ABSTRACT: Burkholderia pseudomallei is a category B agent that causes Melioidosis, an acute and chronic disease with septicemia. The current treatment regimen is a heavy dose of antibiotics such as ceftazidime (CAZ); however, the risk of a relapse is possible. Peptide antibiotics are an alternative to classical antibiotics as they exhibit rapid action and are less likely to result in the development of resistance. The aim of this study was to determine the bactericidal activity against B. pseudomallei and examine the membrane disrupting abilities of the potent antimicrobial peptides: bactenecin, RTA3, BMAP-18 and CA-MA. All peptides exhibited >97% bactericidal activity at 20 µM, with bactenecin having slightly higher activity. Long term time-kill assays revealed a complete inhibition of cell growth at 50 µM bactenecin and CA-MA. All peptides inhibited biofilm formation comparable to CAZ, but exhibited faster kinetics (within 1 h). Bactenecin exhibited stronger binding to LPS and induced perturbation of the inner membrane of live cells. Interaction of bactenecin with model membranes resulted in changes in membrane fluidity and permeability, leading to leakage of dye across the membrane at levels two-fold greater than that of other peptides. Modeling of peptide binding on the membrane showed stable and deep insertion of bactenecin into the membrane (up to 9 Å). We propose that bactenecin is able to form dimers or large ?-sheet structures in a concentration dependent manner and subsequently rapidly permeabilize the membrane, leading to cytosolic leakage and cell death in a shorter period of time compared to CAZ. Bactenecin might be considered as a potent antimicrobial agent for use against B. pseudomallei.

SUBMITTER: Madhongsa K

PROVIDER: S-EPMC3681726 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Antimicrobial action of the cyclic peptide bactenecin on Burkholderia pseudomallei correlates with efficient membrane permeabilization.

Madhongsa Kanjana K Pasan Supaluk S Phophetleb Onanong O Nasompag Sawinee S Thammasirirak Sompong S Daduang Sakda S Taweechaisupapong Suwimol S Lomize Andrei L AL Patramanon Rina R

PLoS neglected tropical diseases 20130613 6

Burkholderia pseudomallei is a category B agent that causes Melioidosis, an acute and chronic disease with septicemia. The current treatment regimen is a heavy dose of antibiotics such as ceftazidime (CAZ); however, the risk of a relapse is possible. Peptide antibiotics are an alternative to classical antibiotics as they exhibit rapid action and are less likely to result in the development of resistance. The aim of this study was to determine the bactericidal activity against B. pseudomallei and ...[more]

PMID: 23785532

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Project description:BackgroundBurkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics.MethodsThe expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared.ResultsProteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative β-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in β-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified.ConclusionsAnalyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.

Project description:Antimicrobial peptides (AMPs) have recently attracted attention as promising antibacterial agents capable of acting against resistant bacterial strains. In this work, an approach was applied, consisting of the conjugation of a peptide related to the sequences of bactenecin 7 (Bac7) and oncocin (Onc112) with the alkyl(triphenyl)phosphonium (alkyl-TPP) fragment in order to improve the properties of the AMP and introduce new ones, expand the spectrum of antimicrobial activity, and reduce the inhibitory effect on the eukaryotic translation process. Triphenylphosphonium (TPP) derivatives of a decapeptide RRIRPRPPYL were synthesized. It was comprehensively studied how the modification of the AMP affected the properties of the new compounds. It was shown that while the reduction in the Bac7 length to 10 a.a. residues dramatically decreased the affinity to bacterial ribosomes, the modification of the peptide with alkyl-TPP moieties led to an increase in the affinity. New analogs with structures that combined a decapeptide related to Bac7 and Onc112-Bac(1-10, R/Y)-and TPP attached to the C-terminal amino acid residue via alkylamide linkers, inhibited translation in vitro and were found to be more selective inhibitors of bacterial translation compared with eukaryotic translation than Onc112 and Bac7. The TPP analogs of the decapeptide related to Bac7 and Onc112 suppressed the growth of both Gram-negative bacteria, similar to Onc112 and Bac7, and Gram-positive ones, similar to alkyl-TPP derivatives, and also acted against some resistant laboratory strains. Bac(1-10, R/Y)-C2-TPP, containing a short alkylamide linker between the decapeptide and TPP, was transferred into the E. coli cells via the SbmA transporter protein. TPP derivatives of the decapeptide Bac(1-10, R/Y) containing either a decylamide or ethylamide linker caused B. subtilis membrane depolarization, similar to alkyl-TPP. The Bac(1-10, R/Y)-C2-TPP analog was proven to be non-toxic for mammalian cells using the MTT test.

Project description:BackgroundAntimicrobial resistance (AMR) poses a major threat to human health. Whole-genome sequencing holds great potential for AMR identification; however, there remain major gaps in accurately and comprehensively detecting AMR across the spectrum of AMR-conferring determinants and pathogens.MethodsUsing 16 wild-type Burkholderia pseudomallei and 25 with acquired AMR, we first assessed the performance of existing AMR software (ARIBA, CARD, ResFinder, and AMRFinderPlus) for detecting clinically relevant AMR in this pathogen. B. pseudomallei was chosen due to limited treatment options, high fatality rate, and AMR caused exclusively by chromosomal mutation (i.e. single-nucleotide polymorphisms [SNPs], insertions-deletions [indels], copy-number variations [CNVs], inversions, and functional gene loss). Due to poor performance with existing tools, we developed ARDaP (Antimicrobial Resistance Detection and Prediction) to identify the spectrum of AMR-conferring determinants in B. pseudomallei.FindingsCARD, ResFinder, and AMRFinderPlus failed to identify any clinically-relevant AMR in B. pseudomallei; ARIBA identified AMR encoded by SNPs and indels that were manually added to its database. However, none of these tools identified CNV, inversion, or gene loss determinants, and ARIBA could not differentiate AMR determinants from natural genetic variation. In contrast, ARDaP accurately detected all SNP, indel, CNV, inversion, and gene loss AMR determinants described in B. pseudomallei (n≈50). Additionally, ARDaP accurately predicted three previously undescribed determinants. In mixed strain data, ARDaP identified AMR to as low as ~5% allelic frequency.InterpretationExisting AMR software packages are inadequate for chromosomal AMR detection due to an inability to detect resistance conferred by CNVs, inversions, and functional gene loss. ARDaP overcomes these major shortcomings. Further, ARDaP enables AMR prediction from mixed sequence data down to 5% allelic frequency, and can differentiate natural genetic variation from AMR determinants. ARDaP databases can be constructed for any microbial species of interest for comprehensive AMR detection.FundingNational Health and Medical Research Council (BJC, EPP, DSS); Australian Government (DEM, ES); Advance Queensland (EPP, DSS).

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Antimicrobial action of the cyclic peptide bactenecin on Burkholderia pseudomallei correlates with efficient membrane permeabilization.

Publications

Antimicrobial action of the cyclic peptide bactenecin on Burkholderia pseudomallei correlates with efficient membrane permeabilization.

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