RELEASE KINETICS OF MONOLITHIC MATRIX COATED WITH OUTER MEMBRANE SYSTEM

The reservoir-monolithic type of the controlled release systems is investigated currently,however,the existing kinetic model could not describe the release process well because the release kinetics is rather complicated.In this paper,a simplified release kinetic model for diffusion-controlled monolithic matrix coated with outer membrane systems is proposed and verified by the experimental data of mercaptopurinum release experiment.It shows that the model can well describe the release mechanism (the relative error is under 3%) when drug loading (C d) is above its solubility limit (C s).At the same time,the release characteristics of special cases (D mD f and D mD f) are discussed theoretically.When D mD f the release rate becomes constant,namely,zero order release,and the release rate is independent of the drug membrane.This result provides the theoretical basis for the system of zero order release as well as how to control the release rate and the amount of drug release.When D mD f,the release rate is dependent on the drug release coefficient in the monolithic matrix,solubility and drug loading but independent of the process in the outer membrane,and it is similar to monolithic matrix type.

Study on Outer Membrane Protein Patterns of Escherichia coli O38,O53 and O75 Isolated from Chickens

[Objective] This study aimed to investigate the outer membrane protein （OMP） patterns of Escherichia coli 038, 053 and 075 isolates from chickens. [Method] Eight pathogenic E. coil isolates with various serotypes were used as experimental materials to extract OMP by using supersonic schizolysis method and Sarcosyl. After SDS-PAGE electrophoresis, OMP patterns of the extracted products were determined based on the OMP model diagram. [Result] OMP of eight E. coil isolates with three serotypes were divided into three patterns, to be specific, 2 075 isolates respectively belonged to OMP-I and OMP-II pattern, 1 053 isolate belonged to OMP-II pattern, and 5 038 isolates belonged to OMP-I and OMP-III pattern. [Conclusion] Experimental results showed that E. coli isolates with the same serotype may belong to completely different OMP patterns, while serologically unrelated isolates may belong to the same OMP pattern. OMP of E. coil isolates with the same serotype may generate genetic differentiation; in addition, OMP of E. coli isolates with different serotypes may have different genetic correlation.

Prokaryotic Expression and Identification of Outer Membrane Protein 2 of Chlamydia trachomatis

Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.

A Mathematical Model for Diffusion-Controlled Monolithic Matrix Coated with outer Membrane System

A release model for diffusion-controlled monolithic matrix coated with outer membrane system is proposed and solved by using the refined double integral method. The calculated results are in satisfactory agreement with the experimental release data. The present model can be well used to describe the release process for all cd/cs values. In addition, the release effects of the monolithic matrix coated with outer membrane system are discussed theoretically.

Development and Evaluation of a MAb-Based ELISA for Detection of Chlamy- dophila pneumoniae Infection with Variable Domain 2 and 3 of the Major Outer Membrane protein

Objective This paper aims to develop a monoclonal antibodies （MAbs）- based ELISA for detecting Chlamydophila pneumoniae （C. pneumonioe） antigens in humans with the variable domains （VD） 2 and 3 of the major outer membrane protein （MOMPvD2-VD~） and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. Methods MOMPvo2-vo3were overexpressed in Escherichia coil and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I （156 patients with typical respiratory illness clinically confirmed before） and Group II （57 healthy donors）. Results In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II ＂healthy donors＂, 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. Conclusion The novel MOMPvD2.VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.

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 mLl）, 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.

Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

Bacterial outer membrane vesicle-based cancer nanovaccines

Tumor vaccines,a type of personalized tumor immunotherapy,have developed rapidly in recent decades.These vaccines evoke tumor antigen-specific T cells to achieve immune recognition and killing of tumor cells.Because the immunogenicity of tumor antigens alone is insufficient,immune adjuvants and nanocarriers are often required to enhance anti-tumor immune responses.At present,vaccine carrier development often integrates nanocarriers and immune adjuvants.Among them,outer membrane vesicles(OMVs)are receiving increasing attention as a delivery platform for tumor vaccines.OMVs are natural nanovesicles derived from Gramnegative bacteria,which have adjuvant function because they contain pathogen associated molecular patterns.Importantly,OMVs can be functionally modified by genetic engineering of bacteria,thus laying a foundation for applications as a delivery platform for tumor nanovaccines.This review summarizes 5 aspects of recent progress in,and future development of,OMV-based tumor nanovaccines:strain selection,heterogeneity,tumor antigen loading,immunogenicity and safety,and mass production of OMVs.

Newly Detected Transmission of bla_(KPC-2) by Outer Membrane Vesicles in Klebsiella Pneumoniae

Objective The prevalence of carbapenem-resistant Klebsiella pneumoniae(CR-KP)is a global public health problem.It is mainly caused by the plasmid-carried carbapenemase gene.Outer membrane vesicles(OMVs)contain toxins and other factors involved in various biological processes,includingβ-lactamase and antibiotic-resistance genes.This study aimed to reveal the transmission mechanism of OMV-mediated drug resistance of Klebsiella(K.)pneumoniae.Methods We selected CR-KP producing K.pneumoniae carbapenemase-2(KPC-2)to study whether they can transfer resistance genes through OMVs.The OMVs of CR-KP were obtained by ultracentrifugation,and incubated with carbapenem-sensitive K.pneumoniae for 4 h.Finally,the carbapenem-sensitive K.pneumoniae was tested for the presence of bla_(KPC-2)resistance gene and its sensitivity to carbapenem antibiotics.Results The existence of OMVs was observed by the electron microscopy.The extracted OMVs had bla_(KPC-2)resistance gene.After incubation with OMVs,bla_(KPC-2)resistance gene was detected in sensitive K.pneumoniae,and it became resistant to imipenem and meropenem.Conclusion This study demonstrated that OMVs isolated from KPC-2-producing CR-KP could deliver bla_(KPC-2)to sensitive K.pneumoniae,allowing the bacteria to produce carbapenemase,which may provide a novel target for innovative therapies in combination with conventional antibiotics for treating carbapenem-resistant Enterobacteriaceae.

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

Research progress on Helicobacter pyloriouter membrane protein

Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density,the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.

Proteomic and Functional Analyses of Outer Membrane Vesicles Secreted by Vibrio splendidus

Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment,exhibiting virulence to a variety of cultured animals.The extracellular products secreted by V.splendidus are crucial to bacterial survival and virulence.In this study,the secretion of outer membrane vesicles(OMVs)by V.splendidus was determined,purified,and morphologically characterized.The protein composition of OMVs was analyzed by proteomic analysis.The results showed that approximately 120 proteins were contained in these OMVs,including outer membrane proteins,flagellins,ABC transporters,protease,and iron regulation proteins,etc.,which were involved in bacterial motility,formation of biofilms and the cell membrane components,and cellular localization based on their structural molecule activity,passive transmembrane transporter activity,channel activity,neurotransmitter receptor activity,extracellular ligand-gated ion channel activity,glutamate receptor activity,ligand-gated ion channel activity,and transmembrane signaling receptor activity.To explore the biological functions of OMVs in V.splendidus,the effects of OMVs on the bacterial adaption to iron limitation,antibiotic,and the coelomic fluid of the Apostichopus japonicus were confirmed.This study is the first time to show that V.splendidus secretes OMVs,and OMVs carry functional proteins that enhance bacterial survival under various stresses.

Immunoproteomic Analysis of Bordetella bronchiseptica Outer Membrane Proteins and Identification of New Immunogenic Proteins

Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine of B. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted from B. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry（MALDI-TOF-MS）, a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins for B. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB（B. bronchiseptica strain isolated from a infected rabbit）. The mortality of mice was 80% compared to 100% of positive controls. The identification of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.

Enhancement of CREC sensitivity to fosfomycin by baicalein through increasing outer membrane permeability

Background:To confirm whether baicalein improves the sensitivity of carbapenem-resistant Escherichia coli(CREC)to fosfomycin by increasing the permeability of bacterial outer membrane in vitro experiments.Methods:The clinically isolated CREC strains were amplified and then divided into three groups including baicalein monotherapy groups,fosfomycin monotherapy groups,and baicalein plus fosfomycin groups,and their minimum inhibitory concentrations(MICs)measurement and interpretation were performed according to CLSI interpretive criteria.To determine bacterial permeability after contact with baicalein,CREC were incubated with fluorescein isothiocyanate(FITC)after pretreatment with blank control without baicalein,with 0.25 MIC of baicalein,and with 0.125 MIC of baicalein,followed by observation of the intrabacterial fluorescence intensity of FITC.In addition,CREC were pretreated with 0.125 MIC of baicalein and with blank control without baicalein followed by measurement of alkaline phosphatase(AKP)leak to determine the change of bacterial permeability.Results:The MIC range in baicalein monotherapy groups was from 128 mg/L to 256 mg/L,and the MIC range in fosfomycin monotherapy groups was from 16 mg/L to 1,024 mg/L,but the MIC range in both combination therapy groups was reduced to 4 mg/L to 64 mg/L.The combination use reduced the MIC of each therapy by 75%-96.88%in all strains,and the fractional inhibitory concentration index(FICI)values less than or equal to 0.5.In the permeability assay,no permeabilization of FITC was observed in the blank groups without baicalein,but the intrabacterial FITC aggregation was observed in the groups of pretreatment with 0.25 MIC of baicalein or 0.25 MIC of baicalein.In the AKP leak assay,the AKP leak was more severe at the groups of coincubation with 0.25 MIC of baicalein than those blank groups without baicalein within the first 6 hours.Conclusion:Our study suggests that baicalein may synergistically enhance the antibacterial effect of fosfomycin by increasing the permeability of CREC outer membrane.

30 and 32 kDa outer membrane proteins of Bordetella pertussis as a modulator on promoting degranulation of mast cells

The correlation between the activities of the outer menbrane proteins （OMPs） of Bordetella pertussis and the lgE-mediated asthma was investigated in the present study, in which the OMPs of B. pertussis and their components were prepared by detergent treatment and chromatography, and the molecular weights of the OMPs components were determined by SDS-PAGE. The amounts of total as well as the ovalbumin （OVA）-specific IgE induced by dead B. pertussis whole bacterial vaccine on guinea pigs were detected by ELISA. Meanwhile, the effect of the OMPs and their components to promote the degranulation of guinea pig mast cells was observed by using the mast cell degranulation test, and ELISA assay was used to measure the histatmine levels in the supematants from the mast cell cultures. Histamine sensitive test was used to demonstrate the effects of the OMPs and their components to increase the histamine lethal sensitivity in mice. It was found that four components with molecular weights of 30, 32, 38 and 69 kDa could be obtained from the OMPs of B. pertussts, and the dead whole bacteria vaccine of B. pertussis had the ability to increase the levels of the total as well as the OVA-specific IgE in sera of guinea pigs. The OMPs and their 30 and 32 kDa components demonstrated significantly enhancing effect on the degranulation of guinea pig mast cells, and the histamine levels in the supematants from the mast cell culture treated with OMPs and their 30 and 32 kDa components were also significantly increased. It is evident that the strong adjuvant activity and the enhancing effect to degranulation of mast cells and the release of histamine of certain outer membrane components of B. pertussis could be demonstrated as revealed by the results of the present study, suggesting the possibility of a close relationship between the infection of vaccination with B. pertussis and the IgE-mediated asthma.

ROLE OF OUTER MEMBRANE PROTEINS IN IMIPENEM DIFFUSION IN PSEUDOMONAS AERUGINOSA

The present study identified the properties of porins in the outer membrane in Pseudomonas aeruginosa,and showed the role of outer membrane in determining imipenem diffusion in Pseudomonas aeruginosa The molecular weight of the major outer membrane protein was analyzed by SDS PAGE The purification of the porins in Pseudomonas aeruginosa was achieved by DEAE ion exchange HPLC The purified outer membrane proteins were reconstituted with phosphatidylcholine and dicetylphosphate into membrane vesicles, and were tested by the liposomes swelling method for the diffusion of imipenem The permeability assay showed that OprC (70 kD), OprD 2 (46kD), and OprE(43 kD) were the channel forming proteins But only OprD 2 was thought to be the likely route of imipenem diffusion

Dynamic shielding of bacterial outer membrane vesicles for safe and efficient chemo-immunotherapy against tumors

Bacterial outer membrane vesicles(OMVs)are potent immunostimulants of regulating the tumor microenvironment(TME)for immunotherapy,and can be used to deliver drugs.However,the severe systemic inflammatory response triggered by OMVs upon intravenous(i.v.)injection has limited their application.Here,we developed a safe and effective strategy by conjugating doxorubicin-loaded serum albumin(SA-DOX,AD)onto the surface of OMVs using a matrix metalloproteinase(MMP)-cleavable peptide linker(cL).This approach enabled the dynamic shielding of OMVs to reduce the systemic side effects while simultaneously enhancing the anti-tumor effects through chemo-immunotherapy.Specifically,the resulting OMV-cL-AD formulation exhibited significantly enhanced accumulation at the tumor site after i.v.administration,facilitated by the SA decoration on the OMVs surface.Subsequently,the shield on the OMV-cL-AD was cleaved by the over-expressed MMP in the TME,leading to the release of both OMVs and AD.This process provided OMV-induced immunotherapy and DOX-induced chemotherapy,resulting in synergistic tumor inhibition.In conclusion,our work demonstrated the potential of OMV-cL-AD as an effective immunochemotherapy strategy that can prolong the survival time of mice without inducing side effects.

Bacterial outer membrane vesicles in the fight against cancer

Bacterial outer membrane vesicles(OMVs)are diminutive vesicles naturally released by Gram-negative bacteria.These vesicles possess distinctive characteristics that attract attention for their potential use in drug administration and immunotherapy in cancer treatment.Therapeutic medicines may be delivered via OMVs directly to the tumor sites,thereby minimizing exposure to healthy cells and lowering the risk of systemic toxicity.Furthermore,the activation of the immune system by OMVs has been demonstrated to facilitate the recognition and elimination of cancer cells,which makes them a desirable tool for immunotherapy.They can also be genetically modified to carry specific antigens,immunomodulatory compounds,and small interfering RNAs,enhancing the immune response to cancerous cells and silencing genes associated with disease progression.Combining OMVs with other cancer treatments like chemotherapy and radiation has shown promising synergistic effects.This review highlights the crucial role of bacterial OMVs in cancer,emphasizing their potential as vectors for novel cancer targeted therapies.As researchers delve deeper into the complexities of these vesicles and their interactions with tumors,there is a growing sense of optimism that this avenue of study will bring positive outcomes and renewed hope to cancer patients in the foreseeable future.

Suppression of host plant defense by bacterial small RNAs packaged in outer membrane vesicles

Noncoding small RNAs(sRNAs)packaged in bacterial outer membrane vesicles(OMVs)function as novel mediators of interspecies communication.While the role of bacterial sRNAs in enhancing virulence is well established,the role of sRNAs in the interaction between OMVs from phytopathogenic bacteria and their host plants remains unclear.In this study,we employ RNA sequencing to characterize differentially pack-aged sRNAs in OMVs of the phytopathogen Xanthomonas oryzae pv.oryzicola(Xoc).Our candidate sRNA(Xosr001)was abundant in OMVs and involved in the regulation of OsJMT1 to impair host stomatal immu-nity.Xoc loads Xosr001 into OMVs,which are specifically ttransferred into the mechanical tissues of rice leaves.Xosr001 suppresses OsJMT1 transcript accumulation in vivo,leading to a reduction in MeJA accu-mulation in rice leaves.Furthermore,the application of synthesized Xosr001 sRNA to the leaves of OsJMT1-HA-OE transgenic line results in the suppression of OsJMT1 expression by Xosr001.Notably,the OsJMT1-HA-OE transgenic line exhibited attenuated stomatal immunity and disease susceptibility upon infection with DXosr001 compared to Xoc.These results suggest that Xosr001 packaged in Xoc OMVs functions to suppress stomatal immunity in rice.