Analysis Of Outer Membrane Proteins Research Articles

Utilizing outer membrane proteins (OMPs) as a vaccine to manage motile Aeromonas septicemia (MAS) in Nile tilapia (Oreochromis niloticus) with evaluating the vaccine immunoprotective effectiveness

AbstractMotile aeromonads are an opportunistic and heterogenic bacteria that cause motile Aeromonas septicemia (MAS) which is accompanied with variety of clinical signs and lesions in fish. It has been found that their outer membrane proteins (OMPs) are highly immunogenic and may be used as a vaccine candidate. The present study was undertaken to evaluate OMP protection against Aeromonas hydrophila (A. hydrophila) strain ATCC 7966 (A5 and A8) and A. hydrophila strain DSM 30,187 (A6). Pathogenicity tests revealed that out of 12 isolated strains, seven were highly pathogenic, three were moderate, and two were avirulent. SDS-PAGE analysis of OMPs showed several protein bands (7–22) of different molecular weights ranging from 14 to 346 kDa. The common protein band was 39 kDa. It seems that pathogenicity and the number of protein bands are related in the highly pathogenic strains. Challenged A. hydrophila for Nile tilapia (O. niloticus) previously actively immunized with OMPs (A4, A5, A6, A8, and A11) showed a significant increase in relative percent survival (RPS) ranging from 50 to 100%. Immunological parameters such as immunoglobulin M (IgM), antioxidants (SOD, GSH, and CAT), and nitric oxide (NO) and expression of pro-inflammatory cytokines, interleukin 1β (IL-1β), and tumor necrosis factor-α (TNFα) showed a significant increase in OMP-immunized fish. Challenged A. hydrophila for O. niloticus previously passively immunized with OMPs (A4, A5, A6, A8, and A11) in fish-raised sera showed a significant increase in RPS (20 to 100%) in the vaccinated groups. It is concluded that OMPs from Aeromonas spp. have been identified as suitable target for protection development, especially in the case of heterogenic bacteria.

Comparative structural and immunological analysis of outer membrane proteins and dermonecrotic toxin in Bordetella bronchiseptica canine isolate

Bordetella bronchiseptica is a pathogen causing respiratory infections in mammals. With the improving understanding of companion animals’ welfare, addressing the side effects of bordetella vaccine gains importance in dogs. Studies on diverse subunit vaccines are actively pursued in humans to safely and effectively control bordetellosis. Therefore, our objective was to develop a canine bordetella vaccine inspired by human vaccine development. We evaluated the immunogenicity of the two bacterial components: the outer membrane proteins (OMPs) and the dermonecrotic toxin (DNT) from a canine isolate of B. bronchiseptica. In-silico analysis identified eight domains of DNT, and Domain 3 was selected as the most promising antigen candidate. Additionally, the OMPs were extracted and examined using SDS-PAGE and Western blot analysis. The distinct immunological characteristic of OMPs and DNT-3 were examined individually and in combination. Gene expression and cytokine production were also evaluated in DH82 cells after stimulation with those antigens. Treatment with OMPs resulted in higher level of Th1 related cytokines, while DNT-3 induced a predominant response associated with Th17 and Th2 in the cytokine production. Synergistic effects were observed exclusively on IL-23, indicating increase of a potential risk of side effects when OMPs and DNT act together. These findings provide valuable insights into the reactogenicity of conventional Bordetella vaccines. Further, the presented preclinical data in this study offer an alternative method of the development for an optimal next-generation Bordetella vaccine for companion animals and humans, replacing the acellular vaccines containing both toxin and protein components.

B-Cell Epitope Mapping of TprC and TprD Variants of Treponema pallidum Subspecies Informs Vaccine Development for Human Treponematoses.

Several recent studies have focused on the identification, functional analysis, and structural characterization of outer membrane proteins (OMPs) of Treponema pallidum (Tp). The Tp species encompasses the highly related pallidum, pertenue, and endemicum subspecies of this pathogen, known to be the causative agents of syphilis, yaws, and bejel, respectively. These studies highlighted the importance of identifying surface-exposed OMP regions and the identification of B-cell epitopes that could be protective and used in vaccine development efforts. We previously reported that the TprC and TprD OMPs of Tp are predicted to contain external loops scattered throughout the entire length of the proteins, several of which show a low degree of sequence variability among strains and subspecies. In this study, these models were corroborated using AlphaFold2, a state-of-the-art protein structure modeling software. Here, we identified B-cell epitopes across the full-length TprC and TprD variants using the Geysan pepscan mapping approach with antisera from rabbits infected with syphilis, yaws, and bejel strains and from animals immunized with refolded recombinant TprC proteins from three syphilis strains. Our results show that the humoral response is primarily directed to sequences predicted to be on surface-exposed loops of TprC and TprD proteins, and that the magnitude of the humoral response to individual epitopes differs among animals infected with various syphilis strains and Tp subspecies. Rather than exhibiting strain-specificity, antisera showed various degrees of cross-reactivity with variant sequences from other strains. The data support the further exploration of TprC and TprD as vaccine candidates.

Inhibitory Effects of Polymyxin B and Human LL-37 on the Flagellin Expression in Vibrio vulnificus.

Vibrio vulnificus, an opportunistic human pathogen responsible for primary septicemia, initiates pathogenesis by attachment to the intestinal epithelial cells for which the motility by the polar flagellum plays an essential role. The proteomic analysis of outer membrane proteins showed that the treatment with the 1/2 minimum inhibitory concentration (MIC) of polymyxin B (a bacterial antimicrobial peptide) led to the reduced production of flagellin (a major component of the polar flagellum). Furthermore, the bacterial motility was inhibited in the presence of 1/2 MIC of polymyxin B. V. vulnificus has six flagellin genes organized into the flaFBA and flaCDE loci. The flaA was found to be expressed higher than flaC, and its expression was significantly decreased by polymyxin B. As well as polymyxin B, the 1/2 MIC of LL-37 (a human intestinal antimicrobial peptide) reduced the expression of flaA. In addition, among four fragments of LL-37, KI-18 and FK-13 containing F17KRIVQRIKDELR29 could lead to the decreased expression of flaA. Because the motility closely relates to virulence of V. vulnificus, the findings obtained herein indicate that LL-37 may reduce the bacterial virulence through inhibition of the motility via the polar flagellum.

β-lactam resistance associated with β-lactamase production and porin alteration in clinical isolates of E. coli and K. pneumoniae.

β-lactam resistance represents a worldwide problem and a serious challenge for antimicrobial treatment. Hence this research was conducted to recognize several mechanisms mediating β-lactam resistance in E. coli and K. pneumoniae clinical isolates collected from Mansoura University hospitals, Egypt. A total of 80 isolates, 45 E. coli and 35 K. pneumoniae isolates, were collected and their antibiotic susceptibility was determined by the Disc diffusion method followed by phenotypic and genotypic detection of extended-spectrum β-lactamases (ESBLs), AmpC β-lactamase, carbapenemase enzymes. The outer membrane protein porins of all isolates were analyzed and their genes were examined using gene amplification and sequencing. Also, the resistance to complement-mediated serum killing was estimated. A significant percentage of isolates (93.8%) were multidrug resistance and showed an elevated resistance to β-lactam antibiotics. The presence of either ESBL or AmpC enzymes was high among isolates (83.75%). Also, 60% of the isolated strains were carbapenemase producers. The most frequently detected gene of ESBL among all tested isolates was blaCTX-M-15 (86.3%) followed by blaTEM-1 (81.3%) and blaSHV-1 (35%) while the Amp-C gene was present in 83.75%. For carbapenemase-producing isolates, blaNDM1 was the most common (60%) followed by blaVIM-1 (35%) and blaOXA-48 (13.8%). Besides, 73.3% and 40% of E. coli and K. pneumoniae isolates respectively were serum resistant. Outer membrane protein analysis showed that 93.3% of E. coli and 95.7% of K. pneumoniae isolates lost their porins or showed modified porins. Furthermore, sequence analysis of tested porin genes in some isolates revealed the presence of frameshift mutations that produced truncated proteins of smaller size. β-lactam resistance in K. pneumoniae and E. coli isolates in our hospitals is due to a combination of β-lactamase activity and porin loss/alteration. Hence more restrictions should be applied on β-lactams usage to decrease the emergence of resistant strains.

Comparative analysis of the main outer membrane proteins of Brucella in the diagnosis of brucellosis

Brucellosis has placed a heavy economic burden on numerous countries and has consumed considerable medical resources worldwide. To improve the specificity and sensitivity of serological methods for diagnosing brucellosis, it is important to develop new diagnostic antigens. Brucella outer membrane proteins(omps) possess good immunogenicity, but there is a scarcity of comparative studies of these proteins in the clinical diagnosis of brucellosis. In this study, six recombinant Brucella outer membrane proteins, omp10, omp16, omp19, omp25, omp31 and BP26, were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera of humans, bovines and goats with brucellosis were analyzed by indirect ELISA using these proteins, lipopolysaccharide(LPS) and Rose Bengale Ag, served as positive-control antigens. In diagnosing human and goat serum, BP26 exhibited the highest diagnostic accuracy of 96.45% and 95.00%, respectively, while omp31 exhibited the strongest ability to detect Brucella in bovine serum with an accuracy of 84.03%. Cross-reaction experiments also confirmed that the diagnostic specificities of omp31 and BP26 were higher than those of the LPS and Rose Bengale Ag antigens. The results of this study indicate that omp31 and BP26 are candidate antigens with high potential application value in the clinical diagnosis of brucellosis.

Extraction and Electrophoretic Analysis of Bacterial Lipopolysaccharides and Outer Membrane Proteins.

Lipopolysaccharides (LPS) (or lipooligosaccharides [LOS], which lack the O-antigen side chains characteristic of LPS), and outer membrane proteins (OMP) are major cell-surface molecules in the outer membrane (OM) of gram-negative bacteria. The LPS is responsible for causing endotoxic shock in infected hosts and, in conjunction with some OMPs, provides protection to the bacterium against host innate immune defenses and attachment to host cells. Electrophoretic analysis can provide valuable information regarding the size, number, and variability of LPS/LOS and OMP components between bacterial strains and mutants, which aids in understanding the basic biology and virulence factors of a particular species. Furthermore, highly purified extracts are normally not required if only electrophoretic analysis is to be done, and various methods have been established for such procedures. Here, we review ameliorated procedures for fast and convenient extraction of LPS/LOS and protein-enriched outer membranes (PEOM) for optimal electrophoretic resolution. Specifically, we will describe the phenol-based micro-method for LPS/LOS extraction, a differential extraction procedure with sodium lauryl sarcosinate for PEOM, and gel preparation for electrophoretic analysis of LPS/LOS samples in detail. Graphic abstract: Workflow for the preparation and analysis of LPS/LOS and PEOM.

How the colonic environment influences Enterohaemorrhagic E. coli outer membrane vesicle production, and the interaction between outer membrane vesicles with human host cells

Enterohaemorrhagic E. coli (EHEC) may instigate bloody diarrhoea and haemolytic uraemic syndrome (HUS) due to Shiga toxin (Stx) production. Stx has been detected within outer membrane vesicles (OMVs), which are membrane-derived nanosized proteoliposomes. During colonisation, EHEC encounters many environmental surroundings such as the presence of bile salts and carbon dioxide (CO2). Here, the influence of different intestinal cues on EHEC OMV production was studied. OMV yield was quantified by densitometric analysis of outer membrane proteins F/C and A, following OMV protein separation by SDS-PAGE. Compared to cultures in Luria broth, higher OMV yields were attained following culture in human cell growth medium and simulated colonic environmental medium, with further increases in the presence of bile salts. Interestingly, lower yields were attained in the presence of T84 cells and CO2. The interaction between OMVs and different human cells was also examined by fluorescence microscopy. Here, OMVs incubated with cells showed internalisation by semi confluent but not fully confluent T84 cell monolayers. OMVs were internalised into the lysosomes in confluent Vero and Caco-2 cells, with Stx being transported to the Golgi and then the Endoplasmic reticulum. OMVs were detected within polarised Caco-2 cells, with no impact on the transepithelial electrical resistance by 24 hours. These results suggest that the colonic environmental factors influences OMV production in vivo. Additionally, results highlight the discrepancies which arise when using different cells lines to examine the intestine. Nevertheless, coupled with Stx, OMVs may serve as tools of EHEC which are involved in HUS development.

Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi.

Ciprofloxacin is the choice treatment for infections caused by Salmonella Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn5 transposon mutants of S. Typhi were screened. S. Typhi zxx::EZ-Tn5 mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn5 transposon insertions were sequenced. Null mutants of interrupted genes, as well as inducible genetic constructs, were produced using site-directed mutagenesis, to corroborate phenotypes. SDS-PAGE and Real-time PCR were used to evaluate the expression of proteins and genes, respectively. Five mutants with increased ciprofloxacin susceptibility were found in the screening. The first confirmed mutant was the glutamine synthetase-coding gene glnA. Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the glnA mutant. Expression of ompF increased four times in the glnA null mutant compared to WT strain. To understand the relationship between the expression of glnA and ompF, a strain with the glnA gene under control of the tetracycline-inducible Ptet promoter was created, to modulate glnA expression. Induction of glnA decreased expression of ompF, at the same time that reduced susceptibility to ciprofloxacin. Expression of sRNA MicF, a negative regulator of OmpF was reduced to one-fourth in the glnA mutant, compared to WT strain. In addition, expression of glnL and glnG genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the glnA mutant. Further studies indicate that deletion of glnG decreases susceptibility to CIP, while deletion of micF gene increases susceptibility CIP. Our findings indicate that glnA inactivation promotes ompF expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.

The impact of the colonic milieu on enterohaemorrhagic E. coli outer membrane vesicle production

Haemolytic uraemic syndrome (HUS) is a complication which may arise upon enterohaemorrhagic E. coli (EHEC) colonisation of the colon. The development of HUS is associated to Shiga toxins (Stxs) release leading to organ damage. It has been found that Stx release can occur within outer membrane vesicles (OMVs). Hence, the effect of different intestinal environment cues on EHEC OMV production was examined. OMVs were purified following EHEC growth in Luria broth (LB), simulated colonic environmental medium (SCEM) with or without bile salts, and in the presence or absence of human cell lines. Yield was quantified through SDS-PAGE and densitometric analysis of outer membrane proteins F/C and A. Furthermore, OMV uptake by HEp-2 and T84 cells was analysed by incubating such cells with OMVs labelled fluorescent lipophilic dye and fluorescence microscopy. Different OMV yields were attained under different conditions, with human cell growth medium and SCEM producing significantly more than cultures grown in LB, with further increases in the presence of bile salts. The presence of HEp-2 cells did not affect OMV yield, yet a lower yield was attained in the presence of T84 cells. These results suggest that colonic environmental factors may influence EHEC OMV production in vivo. OMVs were also shown to be up-taken by both cell types. Such observations with T84 cells suggest that the human colonic epithelium can uptake OMVs. Coupled with Stx, this data may offer a paradigm on how OMVs contribute to Stx translocation across the gut barrier, subsequently leading to HUS.

Analysis of Salmonella enterica Serovar Typhi by Outer Membrane Protein (OMP) Profiling, Random Amplification of Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE)

A number of countries, including developed countries, still have typhoid fever as a major problem resulting in frequent outbreaks. The importance of controlling spread of typhoid fever is well known and necessitates periodic studies to delineate epidemiological relationships. Although phage typing remains to be the preferred conventional method for characterisation of typhoid bacilli, it is of limited use due to prevalence of few predominant phage types in the country like India. Therefore, an effort has been made to assess three molecular methods [Outer Membrane Protein (OMP) Profiling, Random Amplification of Polymorphic DNA (RAPD) and Pulsed Field Gel Electrophoresis (PFGE)] for typing of Salmonella enterica serovar Typhi. 128 Salmonella enterica serovar Typhi isolates were identified using biotyping and serotyping followed by antimicrobial susceptibility testing. These isolates were further subjected to OMP analysis, RAPD and PFGE. PFGE (114 unique clusters) was found to be the most discriminatory method followed by RAPD (94 unique clusters) and OMP profiling (50 unique clusters). Multidrug resistant strains were well discriminated by all three methods used in the study. PFGE still remains the most preferred method for detailed epidemiological investigations. However, random amplification of polymorphic DNA and outer membrane protein profiling can also be considered for molecular discrimination of the isolates in the laboratories lacking high-end facilities.

Quantifying Redox Dynamics of c-Type Cytochromes in a Living Cell Suspension of Dissimilatory Metal-reducing Bacteria.

To quickly and accurately quantify the redox dynamics of c-type cytochromes (c-Cyts) in a living cell suspension, diffuse-transmission UV/visible (DT-UV/Vis) and normal UV/Vis spectroscopy were used to record spectra of c-Cyts in living Shewanella oneidensis MR-1 bacteria. DT-UV/Vis showed a higher absorbance of c-Cyts and lower background compared with normal UV/Vis, because interference from cell surface scattering was removed. The extinction coefficients of oxidized c-Cyts (410 nm) and reduced c-Cyts (419 and 552 nm) were observed. Using this method and the obtained c-Cyts extinction coefficients, the redox transformation kinetics of c-Cyts under anoxic conditions were successfully examined in the presence of various electron acceptors, including 9,10-anthraquinone-2,6-disulfonic acid, Cr(VI), Fe(III) citrate and oxygen. Therefore, the in situ spectral analysis of outer-membrane proteins of intact cells using DT-UV/Vis spectroscopy appears a promising method for investigating microbial metal reduction processes in living cell systems.

Validation of molecular heterogeneity of Fluorescent Pseudomonas spp. and correlation with their potential biocontrol traits against fusarium wilt disease

Fluorescent Pseudomonas (FPs) are major rhizospheric bacteria with a variety of plant growth promotion attributes having potential field applications. The genomic and outer membrane protein (OMP) diversity were valued of 11 shortlisted FPs exhibiting potential biocontrol activity. The taxonomic diversity of isolates was studied using genomic fingerprinting assays—random amplification of polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC), BOX, repetitive extragenic palindromic (REP) and (GTG5), and OMP analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Genomic diversity parameters were calculated: total allelic number, polymorphic information content, expected heterozygosity, dominance, and the Shannon and Simpson indices. All isolates were used to prime tomato seeds and plants were challenge-inoculated with Fusarium oxysporum and the disease incidence was assessed under greenhouse condition. Cluster analyses of RAPD and combined Rep analyses classified isolates into two major clusters. SDS-PAGE analysis of OMP resulted in bands in the range 29–66 kDa. Furthermore, the isolates treated with tomato seeds followed by challenge-inoculation with F. oxysporum under greenhouse conditions showed that the three FPs, M80 (SUB1688209 Seq1 KX570929), M96 (SUB1688209 Seq1 KX570930) and T109 (SUB1688209 Seq1 KX570931) were able to induce systemic resistance. The study examined the possible correlation between strain-specific molecular diversity of FPs and provided an account of their potential biological control activity against F. oxysporum.

Comparative Outer Membrane Protein Analysis of High and Low-Invasive Strains of Cronobacter malonaticus.

Cronobacter are an important group of foodborne pathogens that has been linked to life-threatening infections in both infants and adults. The major infections associated with Cronobacter species are neonatal meningitis, necrotizing enterocolitis, and septicaemia. There are seven species in the Cronobacter genus, of which only three are of clinical importance; Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter turicensis. To date most studies have focussed on C. sakazakii as it is the major species associated with neonatal infections. However, recently C. malonaticus, in particular sequence type 7 (ST7), has been noted as being prevalent in adult infections and therefore warranting further investigation. In this study, eight strains of C. malonaticus ST7, that had been isolated from a wide range of sources and varied in their in vitro virulence, were chosen for proteomic analysis of their outer membrane proteins (OMPs). One-dimensional gel analysis revealed a ~29 kDa size band that was only present in the highly invasive strains. Subsequent mass spectrometric analysis identified several peptides that matched the flagellin protein. The presence of flagellin protein was confirmed in 2D gel spot. Mass spectrometry analysis of total OMPs revealed that the four highly invasive C. malonaticus strains expressed the main flagellum proteins that were absent from the four low invasive strains. These were the flagellar hook protein FlgE, flagellar hook-associated protein 1, flagellar hook-associated protein, flagellin, and flagellar hook-filament junction protein FlgL. This data indicates that C. malonaticus flagellar proteins may have an important role in the organism's invasion properties.

Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

Quantitative proteomic analysis of iron-regulated outer membrane proteins in Aeromonas hydrophila as potential vaccine candidates

The iron-regulated outer membrane protein (OMP) of Aeromonas hydrophila is an effective vaccine candidate, but its intrinsic functional components are largely unknown. In this study, we compared the differentially expressed sarcosine-insoluble fractions of A. hydrophila in iron-limited and normal medium using tandem mass tag labeling-based quantitative proteomics, and identified 91 upregulated proteins including 21 OMPs and 83 downregulated proteins including 10 OMPs. Subsequent bioinformatics analysis showed that iron chelate transport-related proteins were enriched in increasing abundance, whereas oxidoreductase activity and translation-related proteins were significantly enriched in decreasing abundance. The proteomics results were further validated in selected altered proteins by Western blotting. Finally, the vaccine efficacy of five iron-related recombinant OMPs (A0KGW8, A0KFG8, A0KQ46, A0KIU8, and A0KQZ1) that were increased abundance in iron-limited medium, were evaluated when challenged with virulent A. hydrophila against zebrafish, suggesting that these proteins had highly efficient immunoprotectivity. Our results indicate that quantitative proteomics combined with evaluation of vaccine efficacy is an effective strategy for screening novel recombinant antigens for vaccine development.

Colistin resistance associated with outer membrane protein change inKlebsiella pneumoniae and Enterobacter asburiae.

In this study, outer membrane proteins (OMPs) of colistin-resistant Klebsiella pneumoniae and Enterobacter asburiae were analyzed. One colistin-susceptible and three colistin-resistant K. pneumoniae sequence type 258 strains as well as one colistin-susceptible E. asburiae and its colistin-heteroresistant counterpart strain were involved in the study. OMP analysis of each strain was performed by microchip method. Matrix-assisted laser desorption ionization time of flight/mass spectrometry (MALDI-TOF/MS) investigation was carried out after separation of OMPs by two-dimensional gel electrophoresis and in-gel digestion. The MALDI-TOF/MS analysis of OMPs in the colistin-susceptible K. pneumoniae found 16kDa proteins belonging to the LysM domain/BON superfamily, as well as DNA starvation proteins, whereas OmpX and OmpW were detected in the colistin-resistant counterpart strains. OmpC and OmpW were detected in the colistin-susceptible E. asburiae, whereas OmpA and OmpX were identified in the colistin-resistant counterpart. This study demonstrated that OMP differences were between colistin-susceptible and -resistant counterpart strains. The altered Gram-negative cell wall may contribute to acquired colistin resistance in Enterobacteriaceae.

Proteomic Analysis of Outer Membrane Proteins from Salmonella Enteritidis Strains with Different Sensitivity to Human Serum.

Differential analysis of outer membrane composition of S. Enteritidis strains, resistant to 50% normal human serum (NHS) was performed in order to find factors influencing the resistance to higher concentrations of NHS. Ten S. Enteritidis clinical strains, resistant to 50% NHS, all producing very long lipopolysaccharide, were subjected to the challenge of 75% NHS. Five extreme strains: two resistant and three sensitive to 75% NHS, were chosen for the further analysis of outer membrane proteins composition. Substantial differences were found in the levels of particular outer membrane proteins between resistant and sensitive strains, i.e. outer membrane protease E (PgtE) was present mainly in resistant strains, while sensitive strains possessed a high level of flagellar hook-associated protein 2 (FliD) and significantly higher levels of outer membrane protein A (OmpA).

Proteomic analysis of outer membrane proteins and vesicles of a clinical isolate and a collection strain of Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence that displays a high genomic diversity, complicating the study of its pathogenicity, virulence and resistance factors. The interaction of bacterial pathogens with host cells is largely mediated by outer membrane proteins (OMPs). Indeed, several OMPs of Gram-negative bacteria have been recognized as important virulence factors and targets for host immune recognition or to be involved in mechanisms of resistance to antimicrobials. OMPs are also present in outer membrane vesicles (OMVs), which bacteria constitutively secrete to the extracellular milieu and are essential for bacterial survival and pathogenesis. Here, we report the characterization of the OMP and native OMV subproteomes of a clinical isolate (M30) and a collection strain (ATCC13637) of S. maltophilia. We had previously shown that the ATCC13637 strain has an attenuated phenotype in a zebrafish model of infection, as well as a distinct susceptibility profile against a panel of antimicrobials. The protein profiles of the OMP and OMV subproteomes of these two strains and their differences consequently point at pathogenesis, virulence or resistance proteins, such as two variants of the quorum-sensing factor Ax21 that are found to be highly abundant in the OMP fraction and exported to OMVs. Biological significanceStenotrophomonas maltophilia is rapidly climbing positions in the ranking of multidrug-resistant pathogens that are frequently isolated in hospital environments. Being an emerging human pathogen, the knowledge on the factors determining the pathogenicity, virulence and resistance traits of this microorganism is still scarce. Outer membrane proteins (OMPs) and vesicles (OMVs) are key elements for the interaction of Gram-negative bacteria with their environment −including the host–and have fundamental roles in both infection and resistance processes. The present study sets a first basis for a phenotype-dependent characterisation of the OMP subproteome of S. maltophilia and complements very recent work on the OMV subproteome of this species. The variability found among even two strains demonstrates once more that the analysis of genotypically and phenotypically distinct isolates under various conditions will be required before we can draw a significant picture of the OMP and OMV subproteomes of S. maltophilia.

Mechanisms of ertapenem resistance in Enterobacteriaceae isolates in a tertiary university hospital

The aim of this study was to investigate the molecular mechanisms of ertapenem resistance among Enterobacteriaceae isolates in a clinical microbiology laboratory at a tertiary university hospital. A total of...