scholarly journals Colicin E1 Fragments Potentiate Antibiotics by Plugging TolC

2019 ◽  
Author(s):  
S. Jimmy Budiardjo ◽  
Jacqueline J. Deay ◽  
Anna L. Calkins ◽  
Virangika K. Wimalasena ◽  
Daniel Montezano ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Single Molecule ◽  
Outer Membrane Protein ◽  
Efflux Pump ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
E Coli ◽  
Colicin E1 ◽  
Antibiotic Efflux

AbstractThe double membrane architecture of Gram-negative bacteria forms a barrier that is effectively impermeable to extracellular threats. Accordingly, researchers have shown increasing interest in developing antibiotics that target the accessible, surface-exposed proteins embedded in the outer membrane. TolC forms the outer membrane channel of an antibiotic efflux pump in Escherichia coli. Drawing from prior observations that colicin E1, a toxin produced by and lethal to E. coli, can bind to the TolC channel, we investigate the capacity of colicin E1 fragments to ‘plug’ TolC and inhibit its efflux function. First, using single-molecule fluorescence, we show that colicin E1 fragments that do not include the cytotoxic domain localize at the cell surface. Next, using real-time efflux measurements and minimum inhibitory concentration assays, we show that exposure of wild-type E. coli to fragments of colicin E1 indeed disrupts TolC efflux and heightens bacterial susceptibility to four common classes of antibiotics. This work demonstrates that extracellular plugging of outer membrane transporters can serve as a novel method to increase antibiotic susceptibility. In addition to the utility of these protein fragments as starting points for the development of novel antibiotic potentiators, the variety of outer membrane protein colicin binding partners provides an array of options that would allow our method to be used to inhibit other outer membrane protein functions.SignificanceWe find that fragments of a protein natively involved in intraspecies bacterial warfare can be exploited to plug the E. coli outer membrane antibiotic efflux machinery. This plugging disables a primary form of antibiotic resistance. Given the diversity of bacterial species of similar bacterial warfare protein targets, we anticipate that this method of plugging is generalizable to disabling the antibiotic efflux of other proteobacteria. Moreover, given the diversity of the targets of bacterial warfare proteins, this method could be used for disabling the function of a wide variety of other bacterial outer membrane proteins.

scholarly journals The MexJK Efflux Pump of Pseudomonas aeruginosa Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan

2002 ◽  
Vol 184 (18) ◽  
pp. 5036-5044 ◽  
Author(s):  
Rungtip Chuanchuen ◽  
Craig T. Narasaki ◽  
Herbert P. Schweizer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Efflux Pump ◽  
Resistant Mutant ◽  
Efflux System ◽  
Membrane Fusion Protein ◽  
Single Nucleotide Change ◽  
Antibiotic Efflux

ABSTRACT Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.

scholarly journals Molecular Cloning of a Bacteroides caccaeTonB-Linked Outer Membrane Protein Identified by an Inflammatory Bowel Disease Marker Antibody

2001 ◽  
Vol 69 (10) ◽  
pp. 6044-6054 ◽  
Author(s):  
Bo Wei ◽  
Harnisha Dalwadi ◽  
Lynn K. Gordon ◽  
Carol Landers ◽  
David Bruckner ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Bowel Disease ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Enteric Bacteria ◽  
Inflammatory Bowel

ABSTRACT Commensal enteric bacteria are a required pathogenic factor in inflammatory bowel disease (IBD), but the identity of the pertinent bacterial species is unresolved. Using an IBD-associated pANCA monoclonal antibody, a 100-kDa protein was recently characterized from an IBD clinical isolate of Bacteroides caccae (p2Lc3). In this study, consensus oligonucleotides were designed from 100-kDa peptides and used to identify a single-copy gene from the p2Lc3 genome. Sequence analysis of the genomic clone revealed a 2,844-bp (948 amino acid) open reading frame encoding features typical of the TonB-linked outer membrane protein family. This gene, termed ompW,was detected by Southern analysis only in B. caccae and was absent in other species of Bacteroides and gram-negative coliforms. The closest homologues of OmpW included the outer membrane proteins SusC of Bacteroides thetaiotaomicron and RagA of Porphyromonas gingivalis. Recombinant OmpW protein was immunoreactive with the monoclonal antibody, and serum anti-OmpW immunoglobulin A levels were elevated in a Crohn's disease patient subset. These findings suggest that OmpW may be a target of the IBD-associated immune response and reveal its structural relationship to a bacterial virulence factor of P. gingivalis and periodontal disease.

Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenzae : Pathogenic and Epidemiological Implications

1980 ◽  
Vol 30 (3) ◽  
pp. 709-717
Author(s):  
Marilyn R. Loeb ◽  
David H. Smith
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Haemophilus Influenzae ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Protein Composition ◽  
The Other ◽  
Major Protein ◽  
Single Strain

The outer membrane protein composition of 50 disease isolates of Haemophilus influenzae has been determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All strains, including 28 strains of serotype b , one strain each of serotypes a, c, d, e , and f , and 17 untypable strains, had an outer membrane protein composition typical of gram-negative bacteria, i.e., these membranes contained two to three dozen proteins with four to six proteins accounting for most of their protein content. Variation in the mobility of these major outer membrane proteins from strain to strain was common but not universal; the observed patterns provided useful data and new insight into the epidemiology of type b disease. The basic findings can be summarized as follows: (i) All 50 strains possessed three proteins (one minor and two major) each having identical mobilities. The other proteins, both major and minor, varied in mobility. (ii) All type b strains possessed a fourth (major) protein of identical mobility. (iii) The 28 type b strains, on the basis of the mobility of the six major outer membrane proteins, could be divided into eight subtypes. Of all the other strains examined, both typable and untypable, only the serotype a strain belonged to one of these subtypes. (iv) The untypable strains showed considerable variation in the mobilities of their major outer membrane proteins. Of these 17 strains, 13 had an additional major outer membrane protein not present in encapsulated strains. (v) The outer membrane protein composition of a single strain remained unchanged after many passages on solid media, but varied with the growth phase. (vi) The outer membrane protein composition of isolates obtained from nine patients during an epidemic of type b meningitis varied, indicating that a single strain was not responsible for the epidemic. At least five different strains were responsible for these nine cases. (vii) Identical outer membrane protein compositions were observed in the following: in a type b strain and a mutant of this strain deficient in capsule production, indicating that the level of capsule synthesis is not obviously related to outer membrane protein composition; in type b strains isolated from different anatomic sites of patients acutely ill with meningitis, indicating that the strain associated with bacteremia is the same as that isolated from the cerebrospinal fluid; in type b strains isolated from siblings who contracted meningitis at about the same time, indicating infection with the same strain; and in type b strains isolated from the initial and repeat infection of a single patient, suggesting that reinfection was due to the same strain.

BaeR participates in cephalosporins susceptibility by regulating the expression level of outer membrane proteins in Escherichia coli

2020 ◽  
Author(s):  
Shuaiyang Wang ◽  
Chunbo You ◽  
Fareed Qumar Memon ◽  
Geyin Zhang ◽  
Yawei Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Expression Level ◽  
Antibiotics Resistance ◽  
Dilution Method ◽  
Expression Levels ◽  
E Coli

Abstract The two-component system BaeSR participates in antibiotics resistance of Escherichia coli. To know whether the outer membrane proteins involve in the antibiotics resistance mediated by BaeSR, deletion of acrB was constructed and the recombined plasmid p-baeR was introduced into E. coli K12 and K12△acrB. Minimum inhibitory concentrations (MICs) of antibacterial agents were determined by 2-fold broth micro-dilution method. Gene expressions related with major outer membrane proteins and multidrug efflux pump-related genes were determined by real-time quantitative reverse transcription polymerase chain reaction. The results revealed that the MICs of K12ΔacrB to the tested drugs except for gentamycin and amikacin decreased 2- to 16.75-folds compared with those of K12. When BaeR was overexpressed, the MICs of K12ΔacrB/p-baeR to ceftiofur and cefotaxime increased 2.5- and 2-fold, respectively, compared with their corresponding that of K12△acrB. At the same time, the expression levels of ompC, ompF, ompW, ompA and ompX showed significant reduction in K12ΔacrB/p-baeR as compared with K12△acrB. Moreover, the expression levels of ompR, marA, rob and tolC also significantly ‘decreased’ in K12ΔacrB/p-baeR. These findings indicated that BaeR overproduction can decrease cephalosporins susceptibility in acrB-free E. coli by decreasing the expression level of outer membrane proteins.

scholarly journals Sequence Polymorphism, Predicted Secondary Structures, and Surface-Exposed Conformational Epitopes of Campylobacter Major Outer Membrane Protein

2000 ◽  
Vol 68 (10) ◽  
pp. 5679-5689 ◽  
Author(s):  
Qijing Zhang ◽  
Jerrel C. Meitzler ◽  
Shouxiong Huang ◽  
Teresa Morishita
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Major Outer Membrane Protein ◽  
Amino Acid Sequences ◽  
Variable Regions ◽  
Conserved Sequences ◽  
Conformational Epitopes

ABSTRACT The major outer membrane protein (MOMP), a putative porin and a multifunction surface protein of Campylobacter jejuni, may play an important role in the adaptation of the organism to various host environments. To begin to dissect the biological functions and antigenic features of this protein, the gene (designatedcmp) encoding MOMP was identified and characterized from 22 strains of C. jejuni and one strain of C. coli. It was shown that the single-copy cmp locus encoded a protein with characteristics of bacterial outer membrane proteins. Prediction from deduced amino acid sequences suggested that each MOMP subunit consisted of 18 β-strands connected by short periplasmic turns and long irregular external loops. Alignment of the amino acid sequences of MOMP from different strains indicated that there were seven localized variable regions dispersed among highly conserved sequences. The variable regions were located in the putative external loop structures, while the predicted β-strands were formed by conserved sequences. The sequence homology of cmp appeared to reflect the phylogenetic proximity of C. jejuni strains, since strains with identical cmp sequences had indistinguishable or closely related macrorestriction fragment patterns. Using recombinant MOMP and antibodies recognizing linear or conformational epitopes of the protein, it was demonstrated that the surface-exposed epitopes of MOMP were predominantly conformational in nature. These findings are instrumental in the design of MOMP-based diagnostic tools and vaccines.

scholarly journals Purification of the Major Outer Membrane Protein of Azospirillum brasilense, Its Affinity to Plant Roots, and Its Involvement in Cell Aggregation

2001 ◽  
Vol 14 (4) ◽  
pp. 555-561 ◽  
Author(s):  
Saul Burdman ◽  
Gabriella Dulguerova ◽  
Yaacov Okon ◽  
Edouard Jurkevitch
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Azospirillum Brasilense ◽  
Outer Membrane Proteins ◽  
Cell Aggregation ◽  
Major Outer Membrane Protein ◽  
Adhesion Assay ◽  
Fab Fragments

The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

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