scholarly journals Modification of Outer Membrane Protein Profile and Evidence Suggesting an Active Drug Pump in Enterobacter aerogenes Clinical Strains

2003 ◽  
Vol 47 (5) ◽  
pp. 1555-1559 ◽  
Author(s):  
Stéphane Gayet ◽  
Renaud Chollet ◽  
Gérard Molle ◽  
Jean-Marie Pagès ◽  
Jacqueline Chevalier
Keyword(s):  
Outer Membrane ◽  
Protein Profile ◽  
Outer Membrane Proteins ◽  
Enterobacter Aerogenes ◽  
Normal Structure ◽  
Clinical Strains ◽  
Resistance Strategies ◽  
Carbonyl Cyanide ◽  
Energy Dependent ◽  
Drug Pump

ABSTRACT Two clinical strains of Enterobacter aerogenes that exhibited phenotypes of multiresistance to β-lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin were investigated. Both strains showed a porin pattern different from that of a susceptible strain, with a drastic reduction in the amount of the major porin but with an apparently conserved normal structure (size and immunogenicity), together with overproduction of two known outer membrane proteins, OmpX and LamB. In addition, the full-length O-polysaccharide phenotype was replaced by a semirough Ra phenotype. Moreover, in one isolate the intracellular accumulation of chloramphenicol was increased in the presence of the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone, suggesting an energy-dependent efflux of chloramphenicol in this strain. The resistance strategies used by these isolates appear to be similar to that induced by stress in Escherichia coli cells.

scholarly journals Energy-Dependent Accumulation of Fluoroquinolones in Quinolone-Resistant Klebsiella pneumoniaeStrains

1998 ◽  
Vol 42 (7) ◽  
pp. 1850-1852 ◽  
Author(s):  
Luis Martínez-Martínez ◽  
Isabel García ◽  
Sofía Ballesta ◽  
Vicente Javier Benedí ◽  
Santiago Hernández-Allés ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Clinical Isolates ◽  
Intracellular Accumulation ◽  
Active Efflux ◽  
Energy Dependent ◽  
Isogenic Strains

The intracellular accumulation of norfloxacin and pefloxacin inKlebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents.

scholarly journals Protein and antigen profiles of Leptospira interrogans serovar Hardjo

2009 ◽  
Vol 39 (9) ◽  
pp. 2539-2543
Author(s):  
Bárbara Nobre Lafetá ◽  
Elaine Cristina de Castro ◽  
Nivaldo da Silva
Keyword(s):  
Immune Response ◽  
Membrane Proteins ◽  
Western Blot ◽  
Outer Membrane ◽  
Protein Profile ◽  
Outer Membrane Proteins ◽  
Rabbit Antiserum ◽  
Leptospira Interrogans ◽  
Sds Page ◽  
Hyperimmune Rabbit

The protein profile of the outer membrane of Leptospira interrogans serovar Hardjo subtype hardjoprajitno associated with the bovine natural immune response was investigated. The outer membrane proteins were extracted utilizing Triton X114 and precipitated with acetone. The protein sample was then resolved by SDS-PAGE and reacted in western blot against sera from a hyperimmune rabbit and from naturally infected bovines. In silver stained gels, 14 protein bands were observed, among which four proteins, with 22, 29, 47 and 63kDa, appeared as major constituents. Western blot tests with hyperimmune rabbit antiserum detected bands corresponding to proteins with 35; 27; 24; 21; 17 and 14kDa, while 32kDa and 45kDa proteins were the most immunoreactive with sera from naturally infected bovines.

scholarly journals The ner Gene of Photorhabdus: Effects on Primary-Form-Specific Phenotypes and Outer Membrane Protein Composition

2002 ◽  
Vol 184 (11) ◽  
pp. 3096-3105 ◽  
Author(s):  
Keith H. O'Neill ◽  
Declan M. Roche ◽  
David J. Clarke ◽  
Barbara C. A. Dowds
Keyword(s):  
Membrane Protein ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Protein Profile ◽  
Outer Membrane Proteins ◽  
Regulatory Gene ◽  
Phenotypic Switching ◽  
Secondary Form ◽  
Primary Form

ABSTRACT The nematode-bacterium complex of Heterorhabditis-Photorhabdus is pathogenic to insect larvae. The bacteria undergo a form of phenotypic switching whereby the primary form, at the stationary phase of the growth cycle, makes a range of products and has the capacity to support nematode growth, whereas the secondary form does not express these phenotypes. The work described here investigated the mechanism regulating phenotypic variation by transforming the primary cells with secondary-form DNA on a low-copy-number vector and screening for colonies which did not produce the yellow pigment characteristic of primaries. Four transformants all carrying the same gene were found to loose primary-form-specific characteristics, and the gene was sequenced and identified as ner, a regulatory gene in gram-negative bacteria and their phages. Unexpectedly, inactivation of the endogenous gene in the secondaries did not cause them to revert to the primary phenotype, and the gene was expressed in the primary form as well as the secondary form during exponential but not stationary phase and deregulated in the plasmid-bearing primary form. These and other pieces of evidence indicate that the endogenous ner gene is not responsible for the secondary phenotype, but that ner, when overexpressed, can repress expression of primary phenotypes at stationary phase. Inactivation of the endogenous ner gene in the primary form affected the outer membrane protein profile. A number of outer membrane proteins displayed differential accumulation in the primary and secondary forms at stationary phase, and two of the primary-form-specific proteins were absent from the ner primary strain.

scholarly journals Role of TEM-1 β-Lactamase in the Predominance of Ampicillin-Sulbactam-NonsusceptibleEscherichia coliin Japan

2018 ◽  
Vol 63 (2) ◽  
pp. e02366-18
Author(s):  
Taro Noguchi ◽  
Yasufumi Matsumura ◽  
Toru Kanahashi ◽  
Michio Tanaka ◽  
Yasuhiro Tsuchido ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Profile ◽  
Outer Membrane Proteins ◽  
Promoter Sequence ◽  
Resistance Mechanisms ◽  
Wild Type ◽  
Content Type ◽  
E Coli ◽  
Wild Type Promoter

ABSTRACTWe investigated the epidemiology and resistance mechanisms of ampicillin-sulbactam-nonsusceptibleEscherichia coli, focusing on the role of the TEM-1 β-lactamase. We collected all nonduplicateE. coliclinical isolates at 10 Japanese hospitals during December 2014 and examined their antimicrobial susceptibility, β-lactamases, TEM-1 transferability, TEM-1 β-lactamase activity, outer membrane protein profile, membrane permeability, and clonal genotypes. Among the 329 isolates collected, 95 were ampicillin-sulbactam nonsusceptible. Of these ampicillin-sulbactam-nonsusceptible isolates, β-lactamases conferring resistance to sulbactam, such as AmpC, were present in 33%. Hyperproduction of sulbactam-susceptible β-lactamases, TEMs with a strong promoter, were rare (5%). The remaining 59 isolates (62%) had only sulbactam-susceptible β-lactamases, including TEM-1 with a wild-type promoter (n= 28), CTX-Ms (n= 13), or both (n= 17). All 45 transconjugants from 96 donors with TEM-1 had higher ampicillin-sulbactam MICs (4 to 96 mg/liter) than the recipient (2 mg/liter). In donors with only TEM-1, TEM-1 activity correlated with the 50% inhibitory concentration of sulbactam and ampicillin-sulbactam MICs. The decreased membrane permeation of sulbactam was associated with an increased ampicillin-sulbactam MIC. The reduced permeation was partly attributable to deficient outer membrane proteins, which were observed in 57% of the ampicillin-sulbactam-nonsusceptible isolates with only TEM-1 and a wild-type promoter. Sequence type 131 (ST131) was the most common clonal type (52%). TEM-1 with a wild-type promoter primarily contributed to ampicillin-sulbactam nonsusceptibility inE. coli, with the partial support of other mechanisms, such as reduced permeation. Conjugative TEM-1 and the clonal spread of ST131 may contribute to the prevalence of Japanese ampicillin-sulbactam-nonsusceptible isolates.

scholarly journals Surface Loop Motion in FepA

2002 ◽  
Vol 184 (17) ◽  
pp. 4906-4911 ◽  
Author(s):  
Daniel C. Scott ◽  
Salete M. C. Newton ◽  
Phillip E. Klebba
Keyword(s):  
Outer Membrane ◽  
Conformational Changes ◽  
Outer Membrane Proteins ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Ligand Free ◽  
Carbonyl Cyanide ◽  
Surface Loops ◽  
Loop Motion

ABSTRACT Using a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops of Escherichia coli FepA during its transport of the siderophore ferric enterobactin. Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein. The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2. Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332. These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 Å, from a ligand-free open state to a ligand-bound closed state. The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanide m-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide.

scholarly journals Successive Emergence of Enterobacter aerogenes Strains Resistant to Imipenem and Colistin in a Patient

2005 ◽  
Vol 49 (4) ◽  
pp. 1354-1358 ◽  
Author(s):  
Aurélie Thiolas ◽  
Claude Bollet ◽  
Bernard La Scola ◽  
Didier Raoult ◽  
Jean-Marie Pagès
Keyword(s):  
Outer Membrane ◽  
Enterobacter Aerogenes ◽  
Resistance Mechanisms ◽  
Hospital Acquired Infection ◽  
Clinical Strains ◽  
Hospital Acquired

ABSTRACT Enterobacter aerogenes is an agent of hospital-acquired infection that exhibits a remarkable resistance to β-lactam antibiotics during therapy. Five successive isolates of E. aerogenes infecting a patient and exhibiting a multiresistance phenotype to β-lactam antibiotics and fluoroquinolones were investigated. Among these clinical strains, four presented resistant phenotypes during successive imipenem and colistin treatments. The involved resistance mechanisms exhibited by the successive isolates were associated with alterations of the outer membrane that caused a porin decrease and lipopolysaccharide modifications.

scholarly journals Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution ofTreponema pallidumsubsp.pallidum, the syphilis spirochete

2018 ◽  
Author(s):  
Sanjiv Kumar ◽  
Melissa J. Caimano ◽  
Arvind Anand ◽  
Abhishek Dey ◽  
Kelly L. Hawley ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Evolutionary Biology ◽  
Sequence Variation ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum ◽  
Clinical Strains ◽  
Extracellular Loops ◽  
Reference Strains ◽  
Reference Genomes

ABSTRACTIn recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) ofTreponema pallidumsubspeciespallidum(TPA), the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions oftprC,tprD, andbamA, in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we usedtp0548genotyping andtp0558sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants inTPAreference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. Based upon structural models, non-conservative amino acid substitutions in predicted transmembrane β-strands of TprC and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures driveTPAOMP diversity and that genetic exchange contributes to the evolutionary biology ofTPA. They also set the stage for topology-based analysis of antibody responses against OMPs and help frame strategies for syphilis vaccine development.IMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) ofTreponema pallidum(TPA), little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci,tprC,tprD, andbamA,inTPAfrom a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurred predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function also were noted. Our findings suggest that selection pressures exerted by human populations driveTPAOMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance ofTPAand frame strategies for vaccine development based upon conserved OMP extracellular loops.

The Outer Membrane Took Center Stage

2018 ◽  
Vol 72 (1) ◽  
pp. 1-24
Author(s):  
Volkmar Braun
Keyword(s):  
Outer Membrane ◽  
Focal Point ◽  
Outer Membrane Proteins ◽  
Protein Composition ◽  
Structure And Function ◽  
General Interest ◽  
Protein Chemistry ◽  
Iron Chelates ◽  
And Function ◽  
Energy Dependent

My interest in membranes was piqued during a lecture series given by one of the founders of molecular biology, Max Delbrück, at Caltech, where I spent a postdoctoral year to learn more about protein chemistry. That general interest was further refined to my ultimate research focal point—the outer membrane of Escherichia coli—through the influence of the work of Wolfhard Weidel, who discovered the murein (peptidoglycan) layer and biochemically characterized the first phage receptors of this bacterium. The discovery of lipoprotein bound to murein was completely unexpected and demonstrated that the protein composition of the outer membrane and the structure and function of proteins could be unraveled at a time when nothing was known about outer membrane proteins. The research of my laboratory over the years covered energy-dependent import of proteinaceous toxins and iron chelates across the outer membrane, which does not contain an energy source, and gene regulation by iron, including transmembrane transcriptional regulation.

scholarly journals Carbapenem Resistance in a Clinical Isolate of Enterobacter aerogenes Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs

2002 ◽  
Vol 46 (12) ◽  
pp. 3817-3822 ◽  
Author(s):  
Hesna Yigit ◽  
Gregory J. Anderson ◽  
James W. Biddle ◽  
Christine D. Steward ◽  
J. Kamile Rasheed ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Clinical Isolate ◽  
Parent Strain ◽  
Outer Membrane Proteins ◽  
Enterobacter Aerogenes ◽  
Carbapenem Resistance ◽  
The United States ◽  
Polyclonal Antisera ◽  
Imipenem Resistance

ABSTRACT We investigated the mechanism of imipenem resistance in Enterobacter aerogenes strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 μg/ml and the meropenem MIC was 8 μg/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was ≤1 μg/ml and the meropenem MIC was ≤0.25 μg/ml. Cefepime MICs also decreased from 8 to 1 μg/ml; however, the MICs of ceftazidime (≥128 μg/ml), cefoxitin (≥32 μg/ml), and cefotaxime (≥64 μg/ml) remained the same. The β-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain E. aerogenes ATCC 13048 were determined. Strains 810 and 810-REV each produced two β-lactamases with pIs of 8.2 and 5.4. The β-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to Escherichia coli porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of Klebsiella pneumoniae (anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data suggest that imipenem resistance in E. aerogenes 810 is primarily associated with the lack of expression of the analogs of the OmpC (42-kDa) and OmpF (39-kDa) outer membrane proteins, which also results in decreased susceptibility to meropenem and cefepime.

scholarly journals Acquisition of Resistance to Carbapenems in Multidrug-Resistant Clinical Strains of Acinetobacter baumannii: Natural Insertional Inactivation of a Gene Encoding a Member of a Novel Family of β-Barrel Outer Membrane Proteins

2005 ◽  
Vol 49 (4) ◽  
pp. 1432-1440 ◽  
Author(s):  
María A. Mussi ◽  
Adriana S. Limansky ◽  
Alejandro M. Viale
Keyword(s):  
Membrane Proteins ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Signal Sequence ◽  
Outer Membrane Proteins ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Transcriptional Unit ◽  
Clinical Strains ◽  
Insertional Inactivation

ABSTRACT The outer membrane proteins responsible for the influx of carbapenem β-lactam antibiotics in the nonfermentative gram-negative pathogen Acinetobacter baumannii are still poorly characterized. Resistance to both imipenem and meropenem in multidrug-resistant clinical strains of A. baumannii is associated with the loss of a heat-modifiable 29-kDa outer membrane protein, designated CarO. The chromosomal locus containing the carO gene was cloned and characterized from different clinical isolates. Only one carO copy, present in a single transcriptional unit, was found in the A. baumannii genome. The carO gene encodes a polypeptide of 247 amino acid residues with a typical N-terminal signal sequence and a predicted transmembrane β-barrel topology. Its absence from different carbapenem-resistant clinical isolates of A. baumannii resulted from the disruption of carO by distinct insertion elements. The overall data thus support the notion that CarO participates in the influx of carbapenem antibiotics in A. baumannii. Moreover, database searches identified the presence of carO homologs only in species of the genera Acinetobacter, Moraxella, and Psychrobacter, disclosing the existence of a novel family of outer membrane proteins restricted to the family Moraxellaceae of the class γ-Proteobacteria.

Sign in / Sign up

Export Citation Format

Share Document