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MSphere Apr 2023Regulation of porin expression in bacteria is complex and often involves small-RNA regulators. Several small-RNA regulators have been described for Burkholderia...
Regulation of porin expression in bacteria is complex and often involves small-RNA regulators. Several small-RNA regulators have been described for Burkholderia cenocepacia, and this study aimed to characterize the biological role of the conserved small RNA NcS25 and its cognate target, outer membrane protein BCAL3473. The B. cenocepacia genome carries a large number of genes encoding porins with yet-uncharacterized functions. Expression of the porin BCAL3473 is strongly repressed by NcS25 and activated by other factors, such as a LysR-type regulator and nitrogen-depleted growth conditions. The porin is involved in transport of arginine, tyrosine, tyramine, and putrescine across the outer membrane. Porin BCAL3473, with NcS25 as a major regulator, plays an important role in the nitrogen metabolism of B. cenocepacia. Burkholderia cenocepacia is a Gram-negative bacterium which causes infections in immunocompromised individuals and in people with cystic fibrosis. A low outer membrane permeability is one of the factors giving it a high level of innate resistance to antibiotics. Porins provide selective permeability for nutrients, and antibiotics can also traverse the outer membrane by this means. Knowing the properties and specificities of porin channels is therefore important for understanding resistance mechanisms and for developing new antibiotics and could help in overcoming permeability issues in antibiotic treatment.
Topics: Burkholderia cepacia complex; Porins; RNA, Small Untranslated; RNA, Bacterial; Gene Expression Regulation, Bacterial; Biofilms; Gene Deletion; Point Mutation; Base Pairing; Bacterial Outer Membrane Proteins; Biological Transport; Biogenic Amines
PubMed: 36971554
DOI: 10.1128/msphere.00083-23 -
Microbiology Spectrum Jun 2022Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the mechanisms of such resistance in low- and middle-income countries...
Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the mechanisms of such resistance in low- and middle-income countries is limited. This study establishes the genotypic mechanisms of β-lactam resistance by whole-genome sequencing (WGS) in 142 P. aeruginosa clinical isolates recovered from three hospitals in Islamabad and Rawalpindi, Pakistan between 2016 and 2017. Isolates were subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion, and their genomes were assembled from Illumina sequencing data. β-lactam resistance was high, with 46% of isolates resistant to piperacillin-tazobactam, 42% to cefepime, 48% to ceftolozane-tazobactam, and 65% to at least one carbapenem. Twenty-two percent of isolates were resistant to all β-lactams tested. WGS revealed that carbapenem resistance was associated with the acquisition of metallo-β-lactamases (MBLs) or extended-spectrum β-lactamases (ESBLs) in the , , and families, and mutations in the porin gene . These resistance determinants were found in globally distributed lineages, including ST235 and ST664, as well as multiple novel STs which have been described in a separate investigation. Analysis of AST results revealed that acquisition of MBLs/ESBLs on top of porin mutations had an additive effect on imipenem resistance, suggesting that there is a selective benefit for clinical isolates to encode multiple resistance determinants to the same drugs. The strong association of these resistance determinants with phylogenetic background displays the utility of WGS for monitoring carbapenem resistance in P. aeruginosa, while the presence of these determinants throughout the phylogenetic tree shows that knowledge of the local epidemiology is crucial for guiding potential treatment of multidrug-resistant P. aeruginosa infections. Pseudomonas aeruginosa is associated with serious infections, and treatment can be challenging. Because of this, carbapenems and β-lactam/β-lactamase inhibitor combinations have become critical tools in treating multidrug-resistant (MDR) P. aeruginosa infections, but increasing resistance threatens their efficacy. Here, we used WGS to study the genotypic and phylogenomic patterns of 142 P. aeruginosa isolates from the Potohar region of Pakistan. We sequenced both MDR and antimicrobial susceptible isolates and found that while genotypic and phenotypic patterns of antibiotic resistance correlated with phylogenomic background, populations of MDR P. aeruginosa were found in all major phylogroups. We also found that isolates possessing multiple resistance mechanisms had significantly higher levels of imipenem resistance compared to the isolates with a single resistance mechanism. This study demonstrates the utility of WGS for monitoring patterns of antibiotic resistance in P. aeruginosa and potentially guiding treatment choices based on the local spread of β-lactamase genes.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Carbapenems; Genomics; Humans; Imipenem; Microbial Sensitivity Tests; Phylogeny; Porins; Pseudomonas Infections; Pseudomonas aeruginosa; Tazobactam; beta-Lactamases
PubMed: 35638817
DOI: 10.1128/spectrum.00766-22 -
Proceedings of the National Academy of... May 2022In comparison to globular proteins, the spontaneous folding and insertion of β-barrel membrane proteins are surprisingly slow, typically occurring on the order of...
In comparison to globular proteins, the spontaneous folding and insertion of β-barrel membrane proteins are surprisingly slow, typically occurring on the order of minutes. Using single-molecule Förster resonance energy transfer to report on the folding of fluorescently labeled outer membrane protein G we measured the real-time insertion of a β-barrel membrane protein from an unfolded state. Folding events were rare and fast (<20 ms), occurring immediately upon arrival at the membrane. This combination of infrequent, but rapid, folding resolves this apparent dichotomy between slow ensemble kinetics and the typical timescales of biomolecular folding.
Topics: Bacterial Outer Membrane Proteins; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Porins; Protein Conformation, beta-Strand; Protein Folding; Single Molecule Imaging
PubMed: 35549548
DOI: 10.1073/pnas.2121487119 -
Microbiology Spectrum Jun 2022Klebsiella pneumoniae is a pathogen known for its high frequency of antimicrobial resistance. Responses to various environmental stresses during its life can influence...
Klebsiella pneumoniae is a pathogen known for its high frequency of antimicrobial resistance. Responses to various environmental stresses during its life can influence the resistance to antibiotics. Here, we demonstrate the role and mechanism of KbvR regulator in the response to environmental osmotic stress and in the effect of osmotic stress on antimicrobial resistance. The mutant strain exhibited increasing tolerance to high osmotic stress and certain antibiotics, including β-lactams. The expression levels of KbvR and outer membrane porin OmpK36 were upregulated in response to high osmotic stress in the wild type (WT), and the deletion of decreased the expression level of . The membrane permeability of the mutant strain was decreased, which was partly restored through the upregulated expression of OmpK36. The DNA affinity purification sequencing (DAP-seq) and microscale thermophoresis (MST) assay disclosed the binding of KbvR to the promoter of the gene, indicating that KbvR directly and positively regulated the expression of OmpK36. The high osmotic stress increased the susceptibility to β-lactams and the expression of in the WT strain. However, the increased expression and the susceptibility to β-lactams in the mutant strain under high osmotic stress were lower than those of WT. In conclusion, our study has identified that high osmotic stress in the environment influenced the resistance of K. pneumoniae to antibiotics and that the regulation of KbvR with OmpR on the expression of OmpK36 was involved in countering high osmotic stress to change the antimicrobial resistance. Klebsiella pneumoniae is considered a global threat because of the rising prevalence of multidrug-resistant strains and their optimal adaptation to clinical environments and the human host. The sensing and adaption abilities of bacteria to the environmental osmotic stress can change the expression of their outer membrane porins, membrane permeability, and resistance to antibiotics. This study reports that KbvR is a newly found regulator that can be upregulated under high osmotic stress and directly regulate the expression of OmpK36 to change the resistance of K. pneumoniae to β-lactam antibiotics. The results demonstrate how adaptation to high osmotic stress changes the sensitivity of K. pneumoniae to antibiotics. The mechanism can be used to sensitize bacteria to antibiotics and highlight new potential strategies for exploiting shared constraints in governing adaptation to diverse environmental challenges.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Bacterial; Humans; Klebsiella Infections; Klebsiella pneumoniae; Microbial Sensitivity Tests; Osmotic Pressure; Porins; beta-Lactamases; beta-Lactams
PubMed: 35658577
DOI: 10.1128/spectrum.00507-22 -
FEBS Letters Jul 1998Beta-barrel pores are found in outer membrane porins of gram-negative bacteria, bacterial toxins and mitochondrial channels. Apart from the beta-barrel the three groups... (Review)
Review
Beta-barrel pores are found in outer membrane porins of gram-negative bacteria, bacterial toxins and mitochondrial channels. Apart from the beta-barrel the three groups show no close sequence or structural homology but these pores exhibit symmetrical voltage gating when reconstituted into planar lipid bilayers. The structures of several of these are known and many site-directed mutants have been examined. As a result it seems evident that the gating is a common characteristic of these unrelated large pores and is not generated by specialised structures in the pore lumen.
Topics: Bacterial Toxins; Ion Channel Gating; Porins; Protein Conformation
PubMed: 9714531
DOI: 10.1016/s0014-5793(98)00761-3 -
Medecine Sciences : M/S Mar 2004In Gram negative bacteria, hydrophilic antibiotics such as beta-lactams and fluoroquinolons used the bacterial porin channel during their entry. The balance of the porin... (Review)
Review
In Gram negative bacteria, hydrophilic antibiotics such as beta-lactams and fluoroquinolons used the bacterial porin channel during their entry. The balance of the porin expression level and the molecular parameters which govern the molecule diffusion through the pore are important physiological points. Acquired in vivo beta-lactam resistance is often associated with porin loss, and recently clinical resistant strains synthetizing mutated porin have been described. These data highlight both the importance of the channel characteristics and the amino acid residues involved in the drug diffusion process. In addition, several mechanisms, including various repressors or activators as well as molecules inhibiting the pore synthesis or activity, argue for the complexity and plasticity of the bacterial control of porin function. All these aspects play a key role in both membrane permeability and efficiency of the antibiotic resistance process.
Topics: Cell Membrane; Diffusion; Drug Resistance, Bacterial; Gene Expression Regulation; Lactams; Permeability; Porins
PubMed: 15067581
DOI: 10.1051/medsci/2004203346 -
ACS Nano Sep 2016DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane...
DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins' large ionic conductance.
Topics: DNA; Lipid Bilayers; Molecular Dynamics Simulation; Nanotechnology; Porins
PubMed: 27504755
DOI: 10.1021/acsnano.6b03759 -
Nature Methods Apr 2024Precise identification and quantification of amino acids is crucial for many biological applications. Here we report a copper(II)-functionalized Mycobacterium smegmatis...
Precise identification and quantification of amino acids is crucial for many biological applications. Here we report a copper(II)-functionalized Mycobacterium smegmatis porin A (MspA) nanopore with the N91H substitution, which enables direct identification of all 20 proteinogenic amino acids when combined with a machine-learning algorithm. The validation accuracy reaches 99.1%, with 30.9% signal recovery. The feasibility of ultrasensitive quantification of amino acids was also demonstrated at the nanomolar range. Furthermore, the capability of this system for real-time analyses of two representative post-translational modifications (PTMs), one unnatural amino acid and ten synthetic peptides using exopeptidases, including clinically relevant peptides associated with Alzheimer's disease and cancer neoantigens, was demonstrated. Notably, our strategy successfully distinguishes peptides with only one amino acid difference from the hydrolysate and provides the possibility to infer the peptide sequence.
Topics: Nanopores; Amino Acids; Peptides; Amino Acid Sequence; Porins
PubMed: 38443507
DOI: 10.1038/s41592-024-02208-7 -
Communications Biology Oct 2022Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation of structurally related cephalosporins, ceftazidime (CAZ), cefotaxime...
Gram-negative porins are the main entry for small hydrophilic molecules. We studied translocation of structurally related cephalosporins, ceftazidime (CAZ), cefotaxime (CTX) and cefepime (FEP). CAZ is highly active on E. coli producing OmpF (Outer membrane protein F) but less efficient on cells expressing OmpC (Outer membrane protein C), whereas FEP and CTX kill bacteria regardless of the porin expressed. This matches with the different capacity of CAZ and FEP to accumulate into bacterial cells as quantified by LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry). Furthermore, porin reconstitution into planar lipid bilayer and zero current assays suggest permeation of ≈1,000 molecules of CAZ per sec and per channel through OmpF versus ≈500 through OmpC. Here, the instant killing is directly correlated to internal drug concentration. We propose that the net negative charge of CAZ represents a key advantage for permeation through OmpF porins that are less cation-selective than OmpC. These data could explain the decreased susceptibility to some cephalosporins of enterobacteria that exclusively express OmpC porins.
Topics: Cefepime; Cefotaxime; Ceftazidime; Cephalosporins; Chromatography, Liquid; Enterobacteriaceae; Escherichia coli; Lipid Bilayers; Monobactams; Porins; Tandem Mass Spectrometry
PubMed: 36198902
DOI: 10.1038/s42003-022-04035-y -
Journal of Bacteriology Mar 2007Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and...
Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus, A. phagocytophilum needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of A. phagocytophilum has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of L-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of A. phagocytophilum. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel beta-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100 p44 paralogs and go through extensive antigenic variation. The 16-transmembrane-domain beta-strands consist of conserved P44 N- and C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of A. phagocytophilum is incomplete and requires the exogenous acquisition of L-glutamine or L-glutamate for function. Efficient diffusion of L-glutamine across the outer membrane suggests that the porin feeds the Anaplasma TCA cycle and that the relatively large pore size provides Anaplasma with the necessary metabolic intermediates from the host cytoplasm.
Topics: Amino Acid Sequence; Anaplasma phagocytophilum; Antibodies, Monoclonal; Bacterial Outer Membrane Proteins; Chromatography, High Pressure Liquid; Diffusion; Hot Temperature; Molecular Sequence Data; Porins; Protein Structure, Secondary; Proteolipids
PubMed: 17172334
DOI: 10.1128/JB.01548-06