Outer Membrane Protein Patterns Research Articles

Effect of sigma E on carbapenem resistance in OXA-48-producing Klebsiella pneumoniae.

Resistance levels of Gram-negative bacteria producing OXA-48 carbapenemase can vary greatly and some of them can even be categorized as susceptible to imipenem and meropenem according to EUCAST breakpoints. This study aimed to reveal resistance mechanisms leading to varying levels of resistance to carbapenems in Klebsiella pneumoniae with blaOXA-48 submitted to the German National Reference Centre for MDR Gram-negative bacteria. Meropenem-susceptible clinical blaOXA-48-bearing K. pneumoniae isolates were put under gradually increasing selective pressure of meropenem. Clinical isolates and spontaneous meropenem-resistant mutants were whole-genome sequenced with Illumina and Oxford Nanopore Technology. Identified mutations apart from porin mutations were genetically constructed in the original clinical isolates using CRISPR/Cas. Clinical isolates and mutants were analysed for MICs, growth rates and expression of porins on mRNA and protein levels. Mutations associated with meropenem resistance were predominantly found in ompK36, but in some cases ompK36 was unaffected. In two mutants, ISs within the rpoE (sigma factor E; σE) operon were detected, directly in or upstream of rseA. These IS1R elements were then inserted into the same position of the susceptible clinical isolates using CRISPR/Cas. CRISPR-rseA-rseB-rseC mutants showed higher resistance levels to carbapenems and cephalosporins, reduced growth rates and reduced expression of major porins OmpK36 and OmpK35 in quantitative RT-PCR and SDS-PAGE. Enhanced synthesis of σE leads to increased resistance to cephalosporins and carbapenems in clinical K. pneumoniae isolates. This effect could be based upon remodelling of expression patterns of outer membrane proteins. The up-regulated σE stress response also leads to a significant reduction in growth rates.

Outer Membrane Vesicles (OMV)-based and Proteomics-driven Antigen Selection Identifies Novel Factors Contributing to Bordetella pertussis Adhesion to Epithelial Cells

Despite high vaccination coverage world-wide, whooping cough, a highly contagious disease caused by Bordetella pertussis, is recently increasing in occurrence suggesting that novel vaccine formulations targeted at the prevention of colonization and transmission should be investigated. To identify new candidates for inclusion in the acellular formulation, we used spontaneously released outer membrane vesicles (OMV)1 as a potential source of key adhesins. The enrichment of Bvg+ OMV with adhesins and the ability of anti-OMV serum to inhibit the adhesion of B. pertussis to lung epithelial cells in vitro were demonstrated. We employed a proteomic approach to identify the differentially expressed proteins in OMV purified from bacteria in the Bvg+ and Bvg− virulence phases, thus comparing the outer membrane protein pattern of this pathogen in its virulent or avirulent state. Six of the most abundant outer membrane proteins were selected as candidates to be evaluated for their adhesive properties and vaccine potential. We generated E. coli strains singularly expressing the selected proteins and assessed their ability to adhere to lung epithelial cells in vitro. Four out of the selected proteins conferred adhesive ability to E. coli. Three of the candidates were specifically detected by anti-OMV mouse serum suggesting that these proteins are immunogenic antigens able to elicit an antibody response when displayed on the OMV. Anti-OMV serum was able to inhibit only BrkA-expressing E. coli adhesion to lung epithelial cells. Finally, stand-alone immunization of mice with recombinant BrkA resulted in significant protection against infection of the lower respiratory tract after challenge with B. pertussis. Taken together, these data support the inclusion of BrkA and possibly further adhesins to the current acellular pertussis vaccines to improve the impact of vaccination on the bacterial clearance.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases (n=43) were tested against 8 antimicrobial agents and typed by outer membrane protein (OMP) pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin (MICs, ≥16 μg mL−1) and sulfamonomethoxine (MICs, ≥64 μg mL−1), but susceptible to norfloxacin (MICs, ≤0.5 μg mL−1). There was a high incidence of resistance to erythromycin (90.70%) and tylosin (93.02%), while a low incidences of resistance to ciprofloxacin (2.33%), enrofloxacin (2.33%) and florfenicol (4.65%). Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

Analysis of the SDS-PAGE patterns of outer membrane proteins from Escherichia coli strains that have lost the ability to form K1 antigen and varied in the susceptibility to normal human serum

We used SDS-polyacrylamide gel electrophoresis to investigate the outer membrane proteins (OMPs) band composition of 19 Escherichia coli K1 strains that have spontaneously lost the ability to form K1 polysaccharide capsule (E. coli K1−) and demonstrated different degrees of susceptibility to the bactericidal action of normal human serum. Presented results showed that there were differences between E. coli K1− strains in OMPs expressing capacity. The analysis performed on OMPs has not revealed a direct association between the different OMPs band composition and the susceptibility of these strains to the serum.

Identification of Outer Membrane Proteins Altered in Response to UVC-Radiation in Vibrio parahaemolyticus and Vibrio alginolyticus

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240J/m(2)) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.

Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern

BackgroundSalmonella enterica serovar Hadar (S. Hadar) is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments.ResultsThe present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h) and the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE) and LC-MS/MS spectrometry, we compared the proteome of enriched-outer membrane fraction before and after exposure to a magnetic field. A total of 11 proteins, displaying more than a two-fold change, were differentially expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated. These proteins were involved in the integrity of cell envelope (TolB, Pal), in the response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF), in the oxidative stress status (bacterioferritin), in virulence (OmpX, Yfgl) or in motility (FlgE and UspF). Complementary experiments associated the down-regulation of FlgE and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore, the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility in exposed cells. This decrease could be partly associated with the up-regulation of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein, was up-regulated.ConclusionsSMF (200 mT) seems to maintain the cell envelope integrity and to submit the exposed cells to an oxidative stress. Some alterations suggest an increase of the ability of exposed cells to form biofilms.

English

In this study, we incubated four strains of Shigella in seawater microcosms (at room temperature and at 4°C) for eight months and we studied the alteration of their morphologic and outer membranes proteins. The starved cells showed an evolution to the filterable minicells state capable to pass membrane pore size 0.45 µm. In addition, the atomic force micrographs showed a reduction of the cells size and an evolution to coccoid-shapes. Outer membrane proteins patterns of stressed bacteria did not changed too much and these modifications were manifested by the appearance of one new band. Key words: Shigella, seawater, starvation, morphological, outer membrane proteins.

Identification of outer membrane proteins of Vibrio parahaemolyticus and Vibrio alginolyticus altered in response to γ-irradiation or long-term starvation

Vibrio parahaemolyticus and Vibrio alginolyticus were subjected to γ-irradiation (0.5 kGy) or starvation by incubation for 8 months in seawater to study modifications in their outer membrane protein patterns. After treatment, outer membrane protein profiles of starved or γ-irradiated bacteria were found to be altered when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Altered proteins were identified by mass spectrometry (MS and MS/MS) and analyses revealed that OmpU can be considered a starvation stress-induced protein. In addition, expression of OtnA, OmpW, OmpA and peptidoglycan-associated lipoprotein decreased to non-detectable levels in starved cells. Furthermore, MltA-interacting protein MipA appeared under γ-irradiation or starvation conditions. Thus, it can be considered to be a γ-irradiation, long-term starvation stress protein in some vibrios.

Reptiles as a Source of Salmonella O48—Clinically Important Bacteria for Children: The Relationship Between Resistance to Normal Cord Serum and Outer Membrane Protein Patterns

Bacteria of the Salmonella O48 somatic antigen group are clinically important strains causing intestinal dysfunction and diarrhoea, especially in children. The susceptibility of Salmonella O48 strains containing sialic acid (N-acetylneuraminic acid (NeuAc)) in lipopolysaccharide (LPS) to the bactericidal action of normal cord serum (NCS) was determined. The authors' previous results published in Microbial Ecology in 2010 indicated that neither the presence of NeuAc in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of normal human serum (NHS), and that the presence of NeuAc in the LPS structure is not sufficient to block the activation of the alternative pathway of complement in NHS. The current results showed that the tested strains showed various sensitivities also to the bactericidal action of NCS. The authors postulate that the presence of certain outer membrane proteins (OMPs) are characteristic of the resistant and sensitive phenotypes of Salmonella O48 strains. To establish a possible relationship between resistance to NCS and OMPs band patterns, ten Salmonella O48 strains were studied as follows: susceptibility to the bactericidal effect of NCS, the mechanisms of NCS activation and OMP band patterns obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Alterations of Outer Membrane Proteins and Virulence Genes Expression in Gamma-IrradiatedVibrio parahaemolyticusandVibrio alginolyticus

Gamma-irradiation technology sterilizes microorganisms and thereby prevents decay and improves the safety and shelf stability of food products. In this study we treated the foodborne pathogens Vibrio parahaemolyticus and Vibrio alginolyticus with gamma-irradiation (0.5 kGy) to evaluate their adaptative response. Outer membrane protein patterns of irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These modifications were manifested by the appearance and/or disappearance of bands as well as in the expression level of certain proteins. In addition, we searched for the presence of eight Vibrio cholerae virulence genes, toxR, toxS, toxRS, ctxA, zot, ace, toxT, and virulence pathogenicity island (VPI), in the genome of investigated strains. The expression of toxR, toxS, VPI, and ace genes in gamma-irradiated bacteria, studied by reverse transcriptase polymerase chain reaction, was altered. These variations were manifested by an increase and/or a decrease in the expression level of tested virulence genes.

Enzymatic, outer membrane proteins and plasmid alterations of starved Vibrio parahaemolyticus and Vibrio alginolyticus cells in seawater

The marine bacteria Vibrio parahaemolyticus and V. alginolyticus were incubated in seawater for 8 months to evaluate their adaptative responses to starvation. The starved cells showed an altered biochemical and enzymatic profiles, respectively, on Api 20E and Api ZYM systems and an evolution to the filterable minicells state capable to pass membrane pore size 0.45 microm. Outer membrane proteins patterns of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. Plasmids profiles analysis showed that V. alginolyticus ATCC 33787 lost three plasmids, whereas other tested strains conserved their initial profiles.

Studies on Adherence and Outer Membrane Protein of Enteropathogenic Escherichiai coli 0127: H6 and Their Related Plasmids

The following characteristics were found in 20 epidemic strains of enteropathogenic Escherichia coli (EPEC) 0127: H6 isolated from an outbreak of neonatal diarrhea: 1) Absent Vero toxin production; 2) No potential for invasiveness; 3) Possession of 1.5 and 60 Md plasmid identical restriction digest and outer membrane protein (OMP) patterns; 4) Ability of localized adherence to HEp-2, HeLa and FL cells; 5) Capability to cause diarrhea in rabbits with destruction of the ileal microvilli at the areas of bacterial adherence. After elimination of the 60 Md plasmid from EPEC 0127: H6 the 45 and 82 Kd OMPs of the parent strain were lost. These plasmid-cured strains became non-adherent to HEp-2, HeLa and FL cells, and unable to cause diarrhea in rabbits. These results suggest that the pathogenic mechanism of EPEC 0127: H6 induced diarrhea may be related to the genes on a 60 Md plasmid expressed as 45 and 82 Kd OMPs which cause localized adherence to epithelial cells and destruction of ileal microvilli. This damage leads to a marked reduction in absorptive surface area, resulting in diarrhea.

PNPase is a key player in the regulation of small RNAs that control the expression of outer membrane proteins

In this report, we demonstrate that exonucleolytic turnover is much more important in the regulation of sRNA levels than was previously recognized. For the first time, PNPase is introduced as a major regulatory feature controlling the levels of the small noncoding RNAs MicA and RybB, which are required for the accurate expression of outer membrane proteins (OMPs). In the absence of PNPase, the pattern of OMPs is changed. In stationary phase, MicA RNA levels are increased in the PNPase mutant, leading to a decrease in the levels of its target ompA mRNA and the respective protein. This growth phase regulation represents a novel pathway of control. We have evaluated other ribonucleases in the control of MicA RNA, and we showed that degradation by PNPase surpasses the effect of endonucleolytic cleavages by RNase E. RybB was also destabilized by PNPase. This work highlights a new role for PNPase in the degradation of small noncoding RNAs and opens the way to evaluate striking similarities between bacteria and eukaryotes.

Serological and molecular characteristics of Vibrio vulnificus biotype 3: evidence for high clonality

Vibrio vulnificus biotype 3 has been implicated as the causative pathogen of an ongoing disease outbreak that erupted in Israel in 1996. Recent work based on multi-locus sequence typing (MLST) showed that V. vulnificus biotype 3 is genetically homogeneous. The aim of this study was to investigate the existence of subpopulations within this homogeneous biotype by characterizing the surface antigens and analysing the sequence diversity of selected outer-membrane protein (OMP)-encoding genes. Rabbit antisera were prepared against biotype 1, 2 and 3 strains. The results of the slide-agglutination test, dot-blot assay (using fresh and boiled cells), and immunoblotting of lipopolysaccharides (LPS) and OMPs were evaluated. By slide-agglutination and dot-blot assays all biotype 3 strains agglutinated with the selected biotype 3 strain. This homogeneity was supported by immunoblot analysis of the LPS. Analysis of OMP patterns revealed that all three biotypes share a considerable number of common bands that are antigenically related. Cluster analysis of DNA sequence data from selected OMP-encoding genes showed that biotype 3 strains form a genetically distinct and homogeneous clone. The homogeneity of surface antigens and the lack of any sequence diversity among both housekeeping and OMP-encoding genes reaffirms the highly clonal nature of biotype 3 and suggests that it has only recently descended from the parent population of V. vulnificus.

Proteus mirabilis isolates of different origins do not show correlation with virulence attributes and can colonize the urinary tract of mice

Proteus mirabilis has been described as an aetiological agent in a wide range of infections, playing an important role in urinary tract infections (UTIs). In this study, a collection of P. mirabilis isolates obtained from clinical and non-clinical sources was analysed in order to determine a possible correlation between origin, virulence factors and in vivo infectivity. Isolates were characterized in vitro, assessing several virulence properties that had been previously associated with P. mirabilis uropathogenicity. Swarming motility, urease production, growth in urine, outer-membrane protein patterns, ability to grow in the presence of different iron sources, haemolysin and haemagglutinin production, and the presence and expression of diverse fimbrial genes, were analysed. In order to evaluate the infectivity of the different isolates, the experimental ascending UTI model in mice was used. Additionally, the Dienes test and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay were performed to assess the genetic diversity of the isolates. The results of the present study did not show any correlation between distribution of the diverse potential urovirulence factors and isolate source. No significant correlation was observed between infectivity and the origin of the isolates, since they all similarly colonized the urinary tract of the challenged mice. Finally, all isolates showed unique ERIC-PCR patterns, indicating that the isolates were genetically diverse. The results obtained in this study suggest that the source of P. mirabilis strains cannot be correlated with pathogenic attributes, and that the distribution of virulence factors between isolates of different origins may correspond to the opportunistic nature of the organism.

Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract both in children and in adults. Two subpopulations of this organism have been described that differ in 16S rRNA gene sequence and virulence traits. Three 16S rRNA types have been defined. 2-DE followed by protein identification by MS revealed significant differences in the outer membrane protein (OMP) patterns of each M. catarrhalis 16S rRNA type. Approximately 130 features were detected on the 2-DE map of each M. catarrhalis 16S rRNA type. However, only 50 features were expressed by all strains. Furthermore, direct profiling of isolated OMP using MALDI-TOF MS resulted in a characteristic spectral fingerprint for each 16S rRNA type. Fingerprints remained identical when intact cells instead of isolated OMP were analyzed. This finding suggests that the source of desorbed ions is the outer membrane. Based on the fingerprint we were able to assign 18 well-characterized clinical M. catarrhalis isolates to the correct subpopulation. Therefore, MALDI-TOF of intact M. catarrhalis provides a rapid and robust tool for M. catarrhalis strain typing that could be applied in epidemiological studies.

Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IR spectroscopy

Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200-900 cm(-1) region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm(-1)) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.

Immobilization Induces Alterations in the Outer Membrane Protein Pattern ofYersiniaruckeri

We compared the outer membrane protein (OMP) pattern of 2-day-old immobilized Yersinia ruckericells (IC) with that of early (FC24) and late (FC48) stationary-phase planktonic counterparts. Fifty-five OMPs were identified. Principal component analysis discriminated between the protein maps of FC and IC. Some OMPs involved in bacterial adaptation were accumulated by both FC48 and IC but the expression of other proteins was controlled by the sessile mode of growth.

Effect of iron restriction on outer membrane protein composition ofPseudomonas strains studied by conventional and microchip electrophoresis

In this study the outer membrane protein (OMP) composition of six Pseudomonas aeruginosa strains had been analyzed by conventional CE and microchip electrophoresis. Bacterial OMPs are important virulence factors and play a significant role in the pathogenesis of infectious diseases. Changes in their composition might refer to the altered pathogenic properties and antibiotic sensitivity of a certain strain. Pathogenic bacteria invading the human host have to multiplicate under iron-restricted conditions that induce changes in the OMP composition. High-molecular-weight OMPs have to be expressed, which serve as receptors for the iron-siderophore complexes. OMP patterns of bacteria obtained by the two different methods in this study were similar, all major proteins could be detected by both techniques, and the molecular weights showed good correlations, although direct comparison of the peak areas is not straightforward due to the different detection methods (UV and LIF). Changes in OMP composition under iron restriction could be detected, and appearance of a 92 kDa protein in all six P. aeruginosa strains and a 94 kDa protein in the KT 2 strain could be demonstrated. Besides that up- and downregulation of certain proteins could be also detected. The increased separation speed, picoliters of sample consumption, baseline separation achieved more frequently by this method--especially in the high-molecular-weight region--showed the advantages of microchip electrophoresis in the analysis of clinical samples.

Proteomic analysis on the expression of outer membrane proteins of Vibrio alginolyticus at different sodium concentrations

The ability of osmoregulation is crucial to marine pathogens that always face the change of osmotic pressure when they shift between natural marine water-bodies and hosts. Previous studies indicated that the expressional patterns of outer membrane proteins (OMPs) changed when Gram-negative bacteria were transferred in different environments. In the present study, proteomic methodologies were used to investigate the expressional pattern of OMPs of Vibrio alginolyticus, a universal marine pathogen, at different Na(+) concentrations. OmpW, OmpV, and Omp TolC were determined to be osmotic stress responsive proteins. Of the three proteins, importantly, OmpV and OmpW showed distinctly reverse changes to each other, indicating that the two proteins might be the two components varied with changed NaCl concentrations. In addition, our results suggest that closely related species of bacteria with available whole genomic databases should be applied after item microorganism species was used when proteins from a bacterium with unavailable whole genomic information were identified by PMF. Therefore, our results not only expand our knowledge on osmotic stress responsive proteins, but also provide valuable information for strategies on screening of these proteins.