The Mycobacterium tuberculosis Outer Membrane Channel Protein CpnT Confers Susceptibility to Toxic Molecules

ABSTRACTMycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake byM. tuberculosisandMycobacterium bovisBCG. In this study, we found that thecpnTmutant ofM. bovisBCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8- to 32-fold. Furthermore, thecpnTmutant ofM. bovisBCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to controlM. tuberculosisinfections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental forM. bovisBCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, thecpnTgene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinicalM. tuberculosisstrains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria.

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An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1400136111 ◽

2014 ◽

Vol 111 (18) ◽

pp. 6750-6755 ◽

Cited By ~ 61

Author(s):

O. Danilchanka ◽

J. Sun ◽

M. Pavlenok ◽

C. Maueroder ◽

A. Speer ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Outer Membrane ◽

Membrane Channel ◽

Channel Protein ◽

Outer Membrane Channel

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Outer membrane channel protein of an oral pathogen binds human cytokine IL-8

Journal of Oral Microbiology ◽

10.1080/20002297.2017.1325206 ◽

2017 ◽

Vol 9 (sup1) ◽

pp. 1325206

Author(s):

Tuuli Ahlstrand

Keyword(s):

Outer Membrane ◽

Membrane Channel ◽

Oral Pathogen ◽

Channel Protein ◽

Outer Membrane Channel ◽

Human Cytokine

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Role of the Outer Membrane and Porins in Susceptibility of β-Lactamase-Producing Enterobacteriaceae to Ceftazidime-Avibactam

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01585-15 ◽

2015 ◽

Vol 60 (3) ◽

pp. 1349-1359 ◽

Cited By ~ 58

Author(s):

Jean-Marie Pagès ◽

Sabine Peslier ◽

Thomas A. Keating ◽

Jean-Philippe Lavigne ◽

Wright W. Nichols

Keyword(s):

Membrane Permeability ◽

Outer Membrane ◽

Efflux Pump ◽

Enterobacter Aerogenes ◽

Antibacterial Activities ◽

Clinical Isolates ◽

Membrane Pore ◽

Content Type ◽

Porin Proteins ◽

Outer Membrane Permeability

This study examined the activity of the novel antimicrobial combination ceftazidime-avibactam againstEnterobacteriaceaeexhibiting different outer membrane permeability profiles, specifically with or without porins and with or without expression of the main efflux pump (AcrAB-TolC). The addition of the outer membrane permeabilizer polymyxin B nonapeptide increased the antibacterial activities of avibactam alone, ceftazidime alone, and ceftazidime-avibactam against the characterized clinical isolates ofEscherichia coli,Enterobacter aerogenes, andKlebsiella pneumoniae. This enhancement of activities was mainly due to increased passive penetration of compounds since inhibition of efflux by the addition of phenylalanine-arginine β-naphthylamide affected the MICs minimally. OmpF (OmpK35) or OmpC (OmpK36) pores were not the major route by which avibactam crossed the outer membranes ofE. coliandK. pneumoniae. In contrast, Omp35 and Omp36 allowed diffusion of avibactam across the outer membrane ofE. aerogenes, although other diffusion channels for avibactam were also present in that species. It was clear that outer membrane permeability and outer membrane pore-forming proteins play a key role in the activity of ceftazidime-avibactam. Nevertheless, the MICs of ceftazidime-avibactam (with 4 mg/liter avibactam) against the ceftazidime-resistant clinical isolates of the three species ofEnterobacteriaceaestudied were ≤8 mg/liter, regardless of outer membrane permeability changes resulting from an absence of defined porin proteins or upregulation of efflux.

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Overcoming Iron Deficiency of anEscherichia colitonBMutant by Increasing Outer Membrane Permeability

Journal of Bacteriology ◽

10.1128/jb.00340-19 ◽

2019 ◽

Vol 201 (17) ◽

Cited By ~ 10

Author(s):

Nan Qiu ◽

Rajeev Misra

Keyword(s):

Membrane Permeability ◽

Outer Membrane ◽

Membrane Integrity ◽

Receptor Protein ◽

Permeability Barrier ◽

Prolonged Incubation ◽

Content Type ◽

E Coli ◽

Outer Membrane Permeability

ABSTRACTThe intake of certain nutrients, including ferric ion, is facilitated by the outer membrane-localized transporters. Due to ferric insolubility at physiological pH,Escherichia colisecretes a chelator, enterobactin, outside the cell and then transports back the enterobactin-ferric complex via an outer membrane receptor protein, FepA, whose activity is dependent on the proton motive force energy transduced by the TonB-ExbBD complex of the inner membrane. Consequently, ΔtonBmutant cells grow poorly on a medium low in iron. Prolonged incubation of ΔtonBcells on low-iron medium yields faster-growing colonies that acquired suppressor mutations in theyejM(pbgA) gene, which codes for a putative inner-to-outer membrane cardiolipin transporter. Further characterization of suppressors revealed that they display hypersusceptibility to vancomycin, a large hydrophilic antibiotic normally precluded from enteringE. colicells, and leak periplasmic proteins into the culture supernatant, indicating a compromised outer membrane permeability barrier. All phenotypes were reversed by supplying the wild-type copy ofyejMon a plasmid, suggesting thatyejMmutations are solely responsible for the observed phenotypes. The deletion of all known cardiolipin synthase genes (clsABC) did not produce the phenotypes similar to mutations in theyejMgene, suggesting that the absence of cardiolipin from the outer membraneper seis not responsible for increased outer membrane permeability. Elevated lysophosphatidylethanolamine levels and the synthetic growth phenotype withoutpldAindicated that defective lipid homeostasis in theyejMmutant compromises outer membrane lipid asymmetry and permeability barrier to allow enterobactin intake, and that YejM has additional roles other than transporting cardiolipin.IMPORTANCEThe work presented here describes a positive genetic selection strategy for isolating mutations that destabilize the outer membrane permeability barrier ofE. coli. Given the importance of the outer membrane in restricting the entry of antibiotics, characterization of the genes and their products that affect outer membrane integrity will enhance the understanding of bacterial membranes and the development of strategies to bypass the outer membrane barrier for improved drug efficacy.

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The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis

Microbiology ◽

10.1099/mic.0.26902-0 ◽

2004 ◽

Vol 150 (4) ◽

pp. 853-864 ◽

Cited By ~ 82

Author(s):

Claudia Mailaender ◽

Norbert Reiling ◽

Harald Engelhardt ◽

Stefan Bossmann ◽

Stefan Ehlers ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Outer Membrane ◽

Mycobacterium Bovis ◽

Mycobacterium Smegmatis ◽

Drug Susceptibility ◽

Nucleic Acid Binding ◽

Outer Membrane Permeability ◽

Syto 9 ◽

Slow Growing ◽

Hydrophilic Solutes

Porins mediate the diffusion of hydrophilic solutes across the outer membrane of mycobacteria, but the efficiency of this pathway is very low compared to Gram-negative bacteria. To examine the importance of porins in slow-growing mycobacteria, the major porin MspA of Mycobacterium smegmatis was expressed in Mycobacterium tuberculosis and Mycobacterium bovis. Approximately 20 and 35 MspA molecules per μm2 cell wall were observed in M. tuberculosis and M. bovis BCG, respectively, by electron microscopy and quantitative immunoblot experiments. Surface accessibility of MspA in M. tuberculosis was demonstrated by flow cytometry. Glucose uptake was twofold faster, indicating that the outer membrane permeability of M. bovis BCG to small and hydrophilic solutes was increased by MspA. This significantly accelerated the growth of M. bovis BCG, identifying very slow nutrient uptake as one of the determinants of slow growth in mycobacteria. The susceptibility of both M. bovis BCG and M. tuberculosis to zwitterionic β-lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. Furthermore, M. tuberculosis became significantly more susceptible to isoniazid, ethambutol and streptomycin. Fluorescence with the nucleic acid binding dye SYTO 9 was 10-fold increased upon expression of mspA. These results indicated that MspA not only enhanced the efficiency of the porin pathway, but also that of pathways mediating access to large and/or hydrophobic agents. This study provides the first experimental evidence that porins are important for drug susceptibility of M. tuberculosis.

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Outer Membrane Permeability of Cyanobacterium Synechocystis sp. Strain PCC 6803: Studies of Passive Diffusion of Small Organic Nutrients Reveal the Absence of Classical Porins and Intrinsically Low Permeability

Journal of Bacteriology ◽

10.1128/jb.00371-17 ◽

2017 ◽

Vol 199 (19) ◽

Cited By ~ 16

Author(s):

Hikaru Kowata ◽

Saeko Tochigi ◽

Hideyuki Takahashi ◽

Seiji Kojima

Keyword(s):

Membrane Permeability ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Inorganic Ions ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Organic Nutrients ◽

Outer Membrane Permeability ◽

Pcc 6803

ABSTRACT The outer membrane of heterotrophic Gram-negative bacteria plays the role of a selective permeability barrier that prevents the influx of toxic compounds while allowing the nonspecific passage of small hydrophilic nutrients through porin channels. Compared with heterotrophic Gram-negative bacteria, the outer membrane properties of cyanobacteria, which are Gram-negative photoautotrophs, are not clearly understood. In this study, using small carbohydrates, amino acids, and inorganic ions as permeation probes, we determined the outer membrane permeability of Synechocystis sp. strain PCC 6803 in intact cells and in proteoliposomes reconstituted with outer membrane proteins. The permeability of this cyanobacterium was >20-fold lower than that of Escherichia coli. The predominant outer membrane proteins Slr1841, Slr1908, and Slr0042 were not permeable to organic nutrients and allowed only the passage of inorganic ions. Only the less abundant outer membrane protein Slr1270, a homolog of the E. coli export channel TolC, was permeable to organic solutes. The activity of Slr1270 as a channel was verified in a recombinant Slr1270-producing E. coli outer membrane. The lack of putative porins and the low outer membrane permeability appear to suit the cyanobacterial autotrophic lifestyle; the highly impermeable outer membrane would be advantageous to cellular survival by protecting the cell from toxic compounds, especially when the cellular physiology is not dependent on the uptake of organic nutrients. IMPORTANCE Because the outer membrane of Gram-negative bacteria affects the flux rates for various substances into and out of the cell, its permeability is closely associated with cellular physiology. The outer membrane properties of cyanobacteria, which are photoautotrophic Gram-negative bacteria, are not clearly understood. Here, we examined the outer membrane of Synechocystis sp. strain PCC 6803. We revealed that it is relatively permeable to inorganic ions but is markedly less permeable to organic nutrients, with >20-fold lower permeability than the outer membrane of Escherichia coli. Such permeability appears to fit the cyanobacterial lifestyle, in which the diffusion pathway for inorganic solutes may suffice to sustain the autotrophic physiology, illustrating a link between outer membrane permeability and the cellular lifestyle.

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Antibiotic Trapping by Plasmid-Encoded CMY-2 β-Lactamase Combined with Reduced Outer Membrane Permeability as a Mechanism of Carbapenem Resistance in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02459-12 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3941-3949 ◽

Cited By ~ 28

Author(s):

Wil H. F. Goessens ◽

Akke K. van der Bij ◽

Ria van Boxtel ◽

Johann D. D. Pitout ◽

Peter van Ulsen ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Permeability ◽

Outer Membrane ◽

Carbapenem Resistance ◽

Covalent Modification ◽

Content Type ◽

E Coli ◽

Outer Membrane Permeability

ABSTRACTA liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS)Escherichia colistrain, but during admission, a carbapenem-resistant (CR)E. colistrain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CRE. colilacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CRE. colipossessedblaCTX-M-15andblaOXA-1. The CRE. colistrain additionally harboredblaCMY-2and demonstrated a >15-fold increase in β-lactamase activity against nitrocefin, but no hydrolysis of meropenem was detected. However, nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation eitherin vitroorin vivowith meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into variousE. colistrains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of “trapping” by CMY-2 β-lactamase plays a role in carbapenem resistance.

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