Outer Membrane Proteins OmpC Research Articles

Resistance to aztreonam-avibactam among clinical isolates of Escherichia coli is primarily mediated by altered penicillin-binding protein 3 and impermeability

This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25–4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability.

Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles

The secretion of extracellular vesicles (EVs) is a common process in Gram-negative bacteria and can be exploited for biotechnological applications. EVs pose a self-adjuvanting, non-replicative vaccine platform, where membrane and antigens are presented to the host immune system in a non-infectious fashion. The secreted quantity of EVs varies between Gram-negative bacterial species and is comparatively high in the model bacterium E. coli. The outer membrane proteins OmpA and OmpF of the fish pathogen Y. ruckeri have been proposed as vaccine candidates to prevent enteric redmouth disease in aquaculture. In this work, Y.ruckeri OmpA or OmpF were expressed in E. coli and recombinant EVs were isolated. To avoid competition between endogenous E. coli OmpA or OmpF, Y. ruckeri OmpA and OmpF were expressed in E. coli strains lacking ompA, ompF, and in a quadruple knockout strain where the four major outer membrane protein genes ompA, ompC, ompF and lamB were removed. Y.ruckeri OmpA and OmpF were successfully expressed in EVs derived from the E. coli mutants as verified by SDS-PAGE, heat modifiability and proteomic analysis using mass-spectrometry. Transmission electron microscopy revealed the presence of EVs in all E. coli strains, and increased EV concentrations were detected when expressing Y. ruckeri OmpA or OmpF in recombinant EVs compared to empty vector controls as verified by nanoparticle tracking analysis. These results show that E. coli can be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri.

Designing of Multi-Epitope Peptide Vaccine Based on Outer Membrane Proteins OmpF, OmpC, and PgtE of Salmonella entericaTyphi.

Consumption of contaminated water and foods by Salmonella Typhi cause the most common enteric disease known as Typhoid fever in both humans and animals. Despite the existence of various vaccines but infectious diseases remain a major cause of mortality worldwide. Nowadays, in-silico tools design a reliable and stable vaccine to combat such infections. The study aimed to design and evaluate a multi-epitope vaccine based on the outer-membrane proteins of Salmonella Typhi. B-cells and T-cells epitopes were predicted. Predicted epitopes were connected by AAY, KK, and GPGPG linkers. Heparin-Binding Hemagglutinin Adhesin (HBHA) has been attached to the N-terminal of the final vaccine as a potent immune adjuvant. Epitope's antigenicity, allergenicity, immunogenicity, and physicochemical characteristics were defined using in-silico tools. Molecular docking of vaccine-TLR4 was done. ∆G of vaccine-TLR4 is -3.91×104 Kcal mol-1 with 1.93 RMSD. The results indicated protein was stable and non-allergen. In conclusion, the multi-epitope vaccine base on outer membrane proteins of the Salmonella Typhi bacterium might be considered to combat typhoid fever.

Designing of Multi-Epitope Peptide Vaccine Based on Outer Membrane Proteins OmpF, OmpC, and PgtE of Salmonella enterica Typhi

Consumption of contaminated water and foods by Salmonella Typhi cause the most common enteric disease known as Typhoid fever in both humans and animals. Despite the existence of various vaccines but infectious diseases remain a major cause of mortality worldwide. Nowadays, in-silico tools design a reliable and stable vaccine to combat such infections. The study aimed to design and evaluate a multi-epitope vaccine based on the outer-membrane proteins of Salmonella Typhi. B-cells and T-cells epitopes were predicted. Predicted epitopes were connected by AAY, KK, and GPGPG linkers. Heparin-Binding Hemagglutinin Adhesin (HBHA) has been attached to the N-terminal of the final vaccine as a potent immune adjuvant. Epitope’s antigenicity, allergenicity, immunogenicity, and physicochemical characteristics were defined using in-silico tools. Molecular docking of vaccine-TLR4 was done. ∆G of vaccine-TLR4 is -3.91×104 Kcal mol-1 with 1.93 RMSD. The results indicated protein was stable and non-allergen. In conclusion, the multi-epitope vaccine base on outer membrane proteins of the Salmonella Typhi bacterium might be considered to combat typhoid fever.

The tigecycline resistance gene tetX has an expensive fitness cost based on increased outer membrane permeability and metabolic burden in Escherichia coli

Livestock-derived tetX-positive Escherichia coli with tigecycline resistance poses a serious risk to public health. Fitness costs, antibiotic residues, and other tetracycline resistance genes (TRGs) are fundamental in determining the spread of tetX in the environment, but there is a lack of relevant studies. The results of this study showed that both tetO and tetX resulted in reduction in growth and an increased in the metabolic burden of E. coli, but the presence of doxycycline reversed this phenomenon. Moreover, the protection of E. coli growth and metabolism by tetO was superior to that of tetX in the presence of doxycycline, resulting in a much lower competitiveness of tetX-carrying E. coli than tetO-carrying E. coli. The results of RNA-seq showed that the increase in outer membrane proteins (ompC, ompF and ompT) of tetX-carrying E. coli resulted in increased membrane permeability and biofilm formation, which is an important reason for fitness costs. Overall, the increased membrane permeability and metabolic burden of E. coli is the mechanistic basis for the high fitness cost of tetX, and the spread of tetO may limit the spread of tetX. This study provides new insights into the rational use of tetracycline antibiotics to control the spread of tetX.

The roles of membrane permeability and efflux pumps in the toxicity of bisphenol S analogues (2,4-bisphenol S and bis-(3-allyl-4-hydroxyphenyl) sulfone) to Escherichia coli K12

Bisphenol S (BPS) analogues are a group of recently reported emerging contaminants in the environment. Bacteria are important components of food webs. However, the potential risks of BPS analogues in bacteria have not been fully addressed. The toxicity effects and related mechanisms of two BPS analogues with different molecular weights (2,4-bisphenol S (2,4-BPS) and bis-(3-allyl-4-hydroxyphenyl) sulfone (TGSA)) on Escherichia coli K12 were compared. The minimum inhibitory concentration (MIC) of 2,4-BPS in the wild-type of E. coli K12 was lower than that of TGSA. The membrane permeability of the wild-type increased significantly after exposed to the same concentrations (0.5–50 nmol L−1) of 2,4-BPS and TGSA. In addition, 2,4-BPS induced more significant changes in membrane permeability than TGSA. Hormetic effects of 2,4-BPS and TGSA in the wild-type strain were noted in the levels of outer membrane proteins (ompC and ompF), multidrug efflux pump acriflavine resistance B (acrB) and type II topoisomerases. Transcriptomic results indicated these two BPS analogues inhibited the function of ABC transporters. In contrast to TGSA, 2,4-BPS affected DNA replication, tricarboxylic acid cycle, oxidative phosphorylation, and inhibited energy metabolism. Compared with wild-type strain, the ΔacrB mutant strain showed enhanced susceptibility to 2,4-BPS and TGSA with their MICs reduced by 20% and 11%, respectively. Deletion of the acrB affected the growth characteristics and induced stronger oxidative stress than the wild-type strain when exposed to 2,4-BPS or TGSA. The results suggested that 2,4-BPS were more toxic to E. coli K12 than TGSA in the concentration range of 0.5–50 nmol L−1, which was supported by the evidence from their impacts on membrane permeability and efflux pumps.

Comparative Molecular Analysis and Antigenicity Prediction of an Outer Membrane Protein (ompC) of Non-typhoidal Salmonella Serovars Isolated from Different Food Animals in Lagos, Nigeria.

Non-typhoidal Salmonella (NTS) infections occur globally with high morbidity and mortality. The public health challenge caused is exacerbated by increasing rate of antibiotic resistance and absence of NTS vaccine. In this study, we characterized the outer membrane protein C (OmpC) serovars isolated from different food animals and predicted antigenicity. ompC of 27 NTS serovars were amplified by polymerase chain reaction (PCR) and sequenced. Sequence data were analysed and B-cell epitope prediction was done by BepiPred tool. T-cell epitope prediction was done by determining peptide-binding affinities of major histocompatibility complex (MHC) classes I and II using NetMHC pan 2.8 and NetMHC-II pan 3.2, respectively. ompC sequence analysis revealed conserved region among ompCs of Salmonella Serovars. A total of 66.7% of ompCs were stable with instability index value < 40 and molecular weight that ranged from 27 745.47 to 32 714.32 kDa. All ompCs were thermostable and hydrophilic with the exception of S. Pomona (14p) isolate that had ompC with GRAVY value of 0.028 making it hydrophobic. Linear B-cell epitope prediction revealed ability of ompC to elicit humoral immunity. Multiple B-cell epitopes that were exposed and buried were observed on several positions on the ompC sequences. T-cell epitope prediction revealed epitopes with strong binding affinity to MHC-I and -II. Strong binding to human leukocyte antigen (HLA-A) ligands, including HLA-A03:1, HLA-A24:02 and HLA-A26:01 in the case of MHC-I were observed. While binding affinity to H-2 IAs, H-2 IAq and H-2 IAu (H-2 mouse molecules) were strongest in the case of MHC-II. ompCs of NTS serovars isolated from different food animal sources indicated ability to elicit humoral and cell-mediated immunity. Hence, ompCs of NTS serovars are potential candidate for production of NTS vaccines.

Serotype-dependent adhesion of Shiga toxin-producing Escherichia coli to bovine milk fat globule membrane proteins.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens that can cause severe symptoms for humans. Raw milk products are often incriminated as vehicule for human STEC infection. However, raw milk naturally contains molecules, such as the milk fat globule membrane and associated proteins, that could inhibit pathogen adhesion by acting as mimetic ligands. This study aimed to: (i) evaluate the capability of STEC cells to adhere to bovine milk fat globule membrane proteins (MFGMPs), (ii) highlight STEC surface proteins associated with adhesion and (iii) evaluate the variation between different STEC serotypes. We evaluated the physicochemical interactions between STEC and milk fat globules (MFGs) by analyzing hydrophobic properties and measuring the ζ-potential. We used a plate adhesion assay to assess adhesion between MFGMPs and 15 Escherichia coli strains belonging to three key serotypes (O157:H7, O26:H11, and O103:H2). A relative quantitative proteomic approach was conducted by mass spectrometry to identify STEC surface proteins that may be involved in STEC-MFG adhesion. The majority of E. coli strains showed a hydrophilic profile. The ζ-potential values were between -3.7 and - 2.9 mV for the strains and between -12.2 ± 0.14 mV for MFGs. Our results suggest that non-specific interactions are not strongly involved in STEC-MFG association and that molecular bonds could form between STEC and MFGs. Plate adhesion assays showed a weak adhesion of O157:H7 E. coli strains to MFGMPs. In contrast, O26:H11 and O103:H2 serotypes attached more to MFGMPs. Relative quantitative proteomic analysis showed that the O26:H11 str. 21,765 differentially expressed five outer membrane-associated proteins or lipoproteins compared with the O157:H7 str. EDL933. This analysis also found strain-specific differentially expressed proteins, including four O26:H11 str. 21,765-specific proteins/lipoproteins and eight O103:H2 str. PMK5-specific proteins. For the first time, we demonstrated STEC adhesion to MFGMPs and discovered a serotype effect. Several outer membrane proteins-OmpC and homologous proteins, intimin, Type 1 Fimbriae, and AIDA-I-that may be involved in STEC-MFG adhesion were highlighted. More research on STEC's ability to adhere to MFGMs in diverse biological environments, such as raw milk cheeses and the human gastrointestinal tract, is needed to confirm the anti-adhesion properties of the STEC-MFG complex.

The water extract of Aloe vera prevents fluoxetine-induced multiple-drug resistance of E. coli by inhibiting reactive oxygen species formation and membrane permeability

BackgroundThe medication of synthetic chemical is one of the main treatments for depressive disorders. Different lines of evidence reveal that a long-term exposure to anti-depressants, e.g., fluoxetine, is causing multiple-drug resistance (MDR) of gut microbiomes. The MDR bacterial strains in gut pose a threat to intestinal balance and treatment of future microbial infection. Effective strategies are thus in urgent need to prevent the anti-depressant-mediated MDR of gut microbes. PurposeWe aimed to investigate the potential role of Aloe vera (L.) Burm. f. (aloe; Liliaceae family) to prevent MDR of E. coli being co-cultured with fluoxetine. MethodsThe extract of A. vera was co-cultured with E. coli and fluoxetine to analyze the preventive effect of MDR. To figure out the mechanistic action, the formation of reactive oxygen species (ROS) and the expression of key biomarkers, including outer membrane proteins (OmpF and OmpC), superoxidative stress activator (SoxS) and efflux pumps (AcrA/B-TolC), were determined in E. coli being treated with fluoxetine and aloe extract. In addition, the genetic mutation of transcriptional factors of these biomarkers was determined in the fluoxetine-treated E. coli. ResultsThe water extract of A. vera showed considerable potential to reduce the number of fluoxetine-mediated MDR colonies. The extract robustly suppressed the formation of ROS in E. coli. However, thiourea and N-acetylcysteine, two well-known antioxidants, showed no activity in preventing the formation of bacterial MDR. Additionally, A. vera extract directly affected the fluoxetine-triggered early stress response of E. coli and the expression of downstream genes. Meanwhile, A. vera extract was able to inhibit the genetic mutation of SoxR gene in E. coli, as induced by co-cultured with fluoxetine. By fractionation of the aloe extract, the ethanol precipitate, composing mainly polysaccharides, showed robust activity in preventing the fluoxetine-mediated MDR. ConclusionThis study therefore suggested that the extract of A. vera could be an adjuvant agent to combat bacterial MDR during anti-depressant treatment.

Nanoparticles Promote Bacterial Antibiotic Tolerance via Inducing Hyperosmotic Stress Response.

With the rapid development of nanotechnology, nanoparticles (NPs) are widely used in all fields of life. Nowadays, NPs have shown extraordinary antimicrobial activities and become one of the most popular strategies to combat antibiotic resistance. Whether they are equally effective in combating bacterial persistence, another important reason leading to antibiotic treatment failure, remains unknown. Persister cells are a small subgroup of phenotypic drug-tolerant cells in an isogenic bacterial population. Here, various types of NPs are used in combination with different antibiotics to destroy persisters. Strikingly, rather than eradicating persister cells, a wide range of NPs promote the formation of bacterial persistence. It is uncovered by PCR, thermogravimetric analysis, intracellular potassium ion staining, and molecular dynamics simulation that the persister promotion effect is achieved through exerting a hyperosmotic pressure around the cells. Moreover, protein mass spectrometry, fluorescence microscope images, and SDS-PAGE indicate NPs can further hijack cell osmotic regulatory circuits by inducing aggregation of outer membrane protein OmpA and OmpC. These findings question the efficacy of using NPs as antimicrobial agents and raise the possibility that widely used NPs may facilitate the global emergence of bacterial antibiotic tolerance.

Display of Mycobacterium Tuberculosis Binding Peptide on Escherichia Coli Cell Surface

A recombinant expression vector was constructed to fuse the gene of small molecule peptide PK34 isolated from mycobacteria phages with the E. coli outer membrane protein OmpC gene, express recombinant PK34 and histidine labels on the outer membrane using the OmpC own promoter, and establish a surface display system with magnetic beads,and the surface display system was establisted with magnetic beads. The results showed that the peptide PK34 from mycobacteriophage could be displayed on the surface of E. coli by adopting the characteristic of outer membrane protein OmpC. Mycobacterium could be isolated through effective binding of PK34 within the surface display system. This study provides a new insight of the isolation and screening of Mycobacterium from clinical trial in the future.

Recombinant outer membrane protein OmpC induces protective immunity against Aeromonashydrophila infection in Labeorohita

Aeromonashydrophila is an opportunistic pathogen that causes enormous loss to aquaculture industry. The outer membrane proteins of Aeromonas help in bacterium-host interaction, and are considered to be potential vaccine candidates. In the present study, we evaluated immunogenicity and protective efficacy of recombinant OmpC (rOmpC) of A. hydrophila in Indian major carp, Labeorohita. The rOmpC-vaccinated fish produced specific anti-rOmpC antibodies with a significant antibody titer, and the antisera could specifically detect the rOmpC in the cell lysates of Escherichia coli expressing rOmpC and cross-react with different Aeromonas lysates, indicating the suitability of the anti-rOmpC antisera to detect Aeromonas infection. A significant increase was noted in ceruloplasmin level, myeloperoxidase and anti-protease activities in transient and temporal manner the sera of the rOmpC-immunized fish as compared to PBS-control fish. Higher agglutination- and hemolytic activity titers in the anti-rOmpC antisera indicate stimulation of innate immunity. Expression of immune-related genes comprising various acute phase proteins, cytokines and inflammatory response molecules were modulated in the head kidney of rOmpC-immunized L. rohita. While IgM, IL1β, and TLR-22 were significantly up-regulated at early time points (3 h–72 h), the others showed a transient augmentation at both early and later time points (SOD, lysozymes C and G, NKEF-B, C3, CXCa and TNF-α) in the rOmpC-immunized L. rohita in comparison to PBS-injected controls. These data suggest that the rOmpC-induced immune response is temporally regulated to confer immunity. In vivo challenge of the rOmpC-immunized fish with A. hydrophila showed significantly greater survival when compared to PBS-injected control fish. Thus, our results highlight the immunomodulatory role of rOmpC and demonstrate its protective efficacy in L. rohita, along with the use of anti-rOmpC antisera in detecting Aeromonas infections.

LamB, OmpC, and the Core Lipopolysaccharide of Escherichia coli K-12 Function as Receptors of Bacteriophage Bp7.

Bp7 is a T-even phage with a broad host range specific to Escherichia coli, including E. coli K-12. The receptor binding protein (RBP) of bacteriophages plays an important role in the phage adsorption process and determines phage host range, but the molecular mechanism involved in host recognition of phage Bp7 remains unknown. In this study, the interaction between phage Bp7 and E. coli K-12 was investigated. Based on homology alignment, amino acid sequence analysis, and a competitive assay, gp38, located at the tip of the long tail fiber, was identified as the RBP of phage Bp7. Using a combination of in vivo and in vitro approaches, including affinity chromatography, gene knockout mutagenesis, a phage plaque assay, and phage adsorption kinetics analysis, we identified the LamB and OmpC proteins on the surface of E. coli K-12 as specific receptors involved in the first step of reversible phage adsorption. Genomic analysis of the phage-resistant mutant strain E. coli K-12-R and complementation tests indicated that HepI of the inner core of polysaccharide acts as the second receptor recognized by phage Bp7 and is essential for successful phage infection. This observation provides an explanation of the broad host range of phage Bp7 and provides insight into phage-host interactions.IMPORTANCE The RBPs of T4-like phages are gp37 and gp38. The interaction between phage T4 RBP gp37 and its receptors has been clarified by many reports. However, the interaction between gp38 and its receptors during phage adsorption is still not completely understood. Here, we identified phage Bp7, which uses gp38 as an RBP, and provided a good model to study the phage-host interaction mechanisms in an enterobacteriophage. Our study revealed that gp38 of phage Bp7 recognizes the outer membrane proteins (OMPs) LamB and OmpC of E. coli K-12 as specific receptors and binds with them reversibly. HepI of the inner-core oligosaccharide is the second receptor and binds with phage Bp7 irreversibly to begin the infection process. Determining the interaction between the phage and its receptors will help elucidate the mechanisms of phage with a broad host range and help increase understanding of the phage infection mechanism based on gp38.

Resistance mechanisms and molecular epidemiology of carbapenem-nonsusceptible Escherichia coli in Taiwan, 2012-2015.

PurposeThis study aimed to investigate the resistance mechanisms and molecular epidemiology of carbapenem-nonsusceptible Escherichia coli (CnsEC) in Taiwan.Patients and methodsFrom 2012 to 2015, 237 E. coli isolates with minimum inhibitory concentrations of imipenem or meropenem >1 μg/mL were collected in a nationwide surveillance and subjected to polymerase chain reaction (PCR) for carbapenemase, AmpC-type β-lactamase, and extended spectrum β-lactamase (ESBL) genes. We evaluated outer membrane proteins (OmpF and OmpC) loss and conducted multilocus sequence typing and pulsed-field gel electrophoresis (PFGE). Isolates that were resistant to all carbapenems were designated as pan-carbapenem-resistant E. coli (pCREC) in this study.ResultsThe predominant resistance mechanism of CnsEC in Taiwan was the CMY-2 β-lactamase in combination with OmpF and OmpC loss. Sequence type 131 was the most prevalent type (29.2%). Among 237 CnsEC isolates, 106 (44.7%) isolates were pCREC and 18 (7.59%) produced carbapenemase. The prevalence of carbapenemases increased from 6% in 2012 to 11.36% in 2015. Various carbapenemases including KPC-2, IMP-8, NDM-1, NDM-5, VIM-1, OXA-48, and OXA-181 were identified, with NDM-1 being the most common (38.9%) carbapenemase. Comparison between pCREC and non-pCREC among the non-carbapenemase-producing CnsEC isolates revealed SHV, CMY, co-carriage of SHV and CTX-M and concurrent loss of both OmpF and OmpC were more commonly detected in the pCREC group. PFGE revealed no nationwide clonal spread of carbapenemase-producing E. coli.ConclusionNDM-1 was the most common carbapenemase and combination of CMY-2 and concurrent OmpF and OmpC porin loss was the most prevalent resistance mechanism in CnsEC in Taiwan.

Cyclic OmpC peptidic epitope conjugated to tetanus toxoid as a potential vaccine candidate against shigellosis

In earlier works we have described that mice immunized with outer membrane protein OmpC survive the challenge with live Shigella flexnerii 3a. We have also identified conformational epitope of this protein, that was recognized by mice antibodies. The aim of current work was to investigate whether synthetic OmpC epitope homologs can elicit immunological response sufficient in protecting mice against shigellosis. Several linear peptides containing RYDERY motif were synthesized and conjugated to poly-lysine. These conjugates appeared to be poor immunogens and to boost the immunological response an addition of the adjuvant (MPL) was required. Unfortunately, the MPL alone caused a very high immunological reaction that was masking response to peptidic epitope. Under those circumstances we used tetanus toxoid (TT) as the carrier protein for the peptides and the agent stimulating immunological response. Series of cyclic peptides, homologs of the OmpC main epitope were synthesized and conjugated to TT. The loop size in cyclic peptides varied by number of glycine residues, i.e., 1–3 residues added to the GLNRYDERYIGK motif. The linear GLNRYDERYIGC-TT was also prepared as the control. The latter conjugate gave the highest immunological response, followed by the cyclic-GGLNRYDERYIGC-TT and cyclic-GLNRYDERYIGC-TT. The third peptide, cyclic-GGGLNRYDERYIGC-TT, gave a very low response, although it was the most resistant to proteolysis. However, antibodies obtained against cyclic-GGLNRYDERYIGC-TT were more potent to recognize both OmpC and Shigella flexnerii 3a cells than the antibodies against linear GLNRYDERYIGC-TT. Furthermore, the monoclonal antibodies raised against linear GLNRYDERYIGC-TT showed 20-fold lower dissociation constant (KD) than the naturally occurring polyclonal antibodies from umbilical cord sera. Monoclonal antibodies also gave a weaker signal in electron microscope than mice and human polyclonal antibodies. In overall, our results point to cyclic peptides as better candidates for a vaccine development, since they are eliciting production of the higher affinity antibodies against Shigella cells and OmpC.

Th2-biased immune response and agglutinating antibodies generation by a chimeric protein comprising OmpC epitope (323-336) of Aeromonas hydrophila and LTB.

Aeromonas hydrophila is responsible for causing fatal infections in freshwater fishes. Besides chemical/antibiotic treatment and whole-cell vaccine, no subunit vaccine is currently available for A. hydrophila. Outer membrane proteins of gram-negative bacteria have been reported as effective vaccine candidates. Peptide antigens elicit focused immune responses against immunodominant stretches of the antigen. We have attempted to characterize the immunogenicity of linear B-cell epitopes of outer membrane protein (OmpC) of A. hydrophila identified using in silico tools, in conjugation with heat-labile enterotoxin B (LTB) subunit of Escherichia coli as a carrier protein. Antisera against the fusion protein harboring 323-336 residues of the AhOmpC (raised in mice) showed maximum cross-reactivity with the parent protein OmpC and LTB. The fusion protein displayed efficient GM1 ganglioside receptor binding, retaining the adjuvanicity of LTB. Antibody isotype profile and in vitro T-cell response analysis, cytokine ELISA, and array analysis collectively revealed a Th2-biased mixed T-helper cell response. Agglutination assay and flow cytometry analysis validated the ability of anti-fusion protein antisera to recognize the surface exposed epitopes on Aeromonas cells, demonstrating its neutralization potential. Oral immunization studies in Labeo rohita resulted in the generation of long-lasting humoral immune response, and the antisera could cross-react with the fusion protein as well as both the fusion partners. Considering significant similarity among OmpC of different enteric bacteria, the use of A. hydrophila OmpC epitope323-336 in fusion with LTB could have a broader scope in vaccine design.

Salmonella Typhi Porins OmpC and OmpF Are Potent Adjuvants for T-Dependent and T-Independent Antigens.

Several microbial components, such as bacterial DNA and flagellin, have been used as experimental vaccine adjuvants because of their inherent capacity to efficiently activate innate immune responses. Likewise, our previous work has shown that the major Salmonella Typhi (S. Typhi) outer membrane proteins OmpC and OmpF (porins) are highly immunogenic protective antigens that efficiently stimulate innate and adaptive immune responses in the absence of exogenous adjuvants. Moreover, S. Typhi porins induce the expression of costimulatory molecules on antigen-presenting cells through toll-like receptor canonical signaling pathways. However, the potential of major S. Typhi porins to be used as vaccine adjuvants remains unknown. Here, we evaluated the adjuvant properties of S. Typhi porins against a range of experimental and clinically relevant antigens. Co-immunization of S. Typhi porins with ovalbumin (OVA), an otherwise poorly immunogenic antigen, enhanced anti-OVA IgG titers, antibody class switching, and affinity maturation. This adjuvant effect was dependent on CD4+ T-cell cooperation and was associated with an increase in IFN-γ, IL-17A, and IL-2 production by OVA-specific CD4+ T cells. Furthermore, co-immunization of S. Typhi porins with an inactivated H1N1 2009 pandemic influenza virus experimental vaccine elicited higher hemagglutinating anti-influenza IgG titers, antibody class switching, and affinity maturation. Unexpectedly, co-administration of S. Typhi porins with purified, unconjugated Vi capsular polysaccharide vaccine (Vi CPS)—a T-independent antigen—induced higher IgG antibody titers and class switching. Together, our results suggest that S. Typhi porins OmpC and OmpF are versatile vaccine adjuvants, which could be used to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and T-independent antigens.

RpoE is a Putative Antibiotic Resistance Regulator of Salmonella enteric Serovar Typhi.

Bacterial antimicrobial resistance has been associated with the up regulation of genes encoding efflux pumps and the down regulation of genes encoding outer membrane proteins (OMPs). Gene expression in bacteria is primarily initiated by sigma factors (σ factors) such as RpoE, which plays an important role in responding to many environmental stresses. Here, we report the first observation that RpoE serves as an antibiotic resistance regulator in Salmonella enteric serovar Typhi (S. Typhi). In this study, we found that the rpoE mutant (ΔrpoE) of S. Typhi GIFU10007 has elevated resistance to several antimicrobial agents, including β-lactams, quinolones, and aminoglycosides. Genomic DNA microarray analysis was used to investigate the differential gene expression profiles between a wild type and rpoE mutant in response to ampicillin. The results showed that a total of 57 genes displayed differential expression (two-fold increase or decrease) in ΔrpoE versus the wild-type strain. The expressions of two outer membrane protein genes, ompF and ompC, were significantly down-regulated in ΔrpoE (six and seven-fold lower in comparison to wild-type strain) and RamA, a member of the efflux pump AraC/XylS family, was up-regulated about four-fold in the ΔrpoE. Our results suggest RpoE is a potential antimicrobial regulator in S. Typhi, controlling both the down regulation of the OMP genes and up-regulating the efflux system.

Impact of Outer Membrane Protein OmpC and OmpF on Antibiotics Resistance of E. coli Isolated from UTI and Diarrhoeic Patients in Zaria, Nigeria

Multidrug resistant (MDR) E. coli associated infections remains one of the most important bacterial infections that have contributed significantly to increased morbidity and mortality in clinical settings. One of the known resistant mechanisms of MDR bacteria is reduced cell wall permeability, which is controlled by outer membrane protein OmpF and OmpC. This study evaluates the difference in molecular weight of outer membrane protein of MDR E. coli isolated from UTI and diarrhoeic patients in Zaria, Nigeria and antibiotic susceptible ATCC29522 strain using standard microbiological and molecular techniques. Eighty seven (87) confirmed E. coli isolates from UTI and diarrhoeic patients in selected hospitals in Zaria, Nigeria were evaluated for MDR using 15 antibiotics commonly prescribed for E. coli associated infections. The results showed that the 21 suspected multidrug isolates were 100% susceptible to Imipenem and Amikacin, and 71.4 % susceptible to Nitrofurantoin but highly (100%) resistant to Amoxicillin, Ofloxacin, Ciprofloxacin, Cefpodoxime and Ceftaxime, 95.2% resistant to Cefpirome, 85.7% to Tetracycline and Sulphamethonidazole-Trimethroprim, 76.2% to Gentamicine, 66.7% to Chloramphenicol, 61.9% to Aztreonam and 57.1% to Ceftriaxone. Cell wall protein evaluation using SDS-PAGE showed that both the MDR isolates and susceptible strain had equal OmpC bands at 38kDa while the OmpF varied from one MDR isolate to another compared with the ATCC29522 used as control. This study contributes to other findings that a decrease in cell wall outer protein OmpF could contribute to high resistance to antibiotics.

Similarities between Exogenously- and Endogenously-Induced Envelope Stress: The Effects of a New Antibacterial Molecule, TPI1609-10

Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10's mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.