OBJECTIVETo evaluate the biological characteristics of Campylobacter jejuni (CJ) cultured on different culture media and their expression abundance of outer membrane proteins (OMPs).

METHODSCJ was cultured on the improved Bull's medium yolk agar, improved Bull's blood agar or improved Bull's agar for 48 h. The biological characteristics of the bacteria, including the colony feature, morphology, motility, biochemistry, and results of indirect fluorescence test were observed and compared. OMP of the cultured CJ was extracted using 0.2 mol/L and glycine-hydrochloride buffered solution (pH 2.2) and identified by SDS-PAGE to compare the expression abundance of the OMPs with molecular weight of 28-31 kD.

RESULTSCJ exhibited typical biological characteristics with larger cell body and more rapid growth on improved Bull's medium yolk agar than those on improved Bull's blood agar and improved Bull's agar. The bacteria grown on improved Bull's medium yolk agar showed also greater expression abundance of the OMPs with molecule mass between 28 kD and 31 kD.

CONCLUSIONImproved Bull's medium yolk agar allows rapid growth of CJ with typical biological characteristics and enhanced expression of the OMPs with molecular weight of 28 -31 kD, and can be widely used in CJ subunit vaccine development, CJ epidemiological survey, CJ food safety examination, and CJ quarantine.

Bacterial Outer Membrane Proteins ; analysis ; metabolism ; Campylobacter jejuni ; growth & development ; metabolism ; Culture Media

OBJECTIVETo investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).

METHODSMutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.

RESULTSWhen grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.

CONCLUSIONYpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Bacterial Outer Membrane Proteins ; secretion ; Bacterial Secretion Systems ; genetics ; Genes, Bacterial ; Plasmids ; Protein Interaction Mapping ; Yersinia pestis ; genetics ; metabolism ; pathogenicity

In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa (PA)-induced pulmonary infection, pulmonary infection models were established by intratracheal injection of K767 (wild type), nalB (MexA-MexB-OprM up-regulated mutant), and ΔmexB (knockout) strains, separately. All mice were treated with Meropenem (intraper Δ itoneal injection, 100 mg/kg body weight, twice every day), and strain-related pathology, bacteria count, cytokine level, myeloperoxidase (MPO, indicator of neutrophil recruitment) activity, and macrophage inflammatory protein-2 (MIP-2) expression were evaluated at early (3rd day post-infection) and late (7th and 14th day post-infection) stages of infection. E-test showed that ΔmexB was more significantly Δ sensitive to panipenan (ETP), meropenem (MP) and imipenem (IP) than K767 and nalB strains. There was no significant difference in sensitivity to cefepime (TM) among the three stains. In contrast to the K767 and nalB groups, the ΔmexB group showed decreased bacteria burden over time and less exte Δ nsive pathological change. Additionally, MPO activity and levels of inflammatory cytokines (IL-1b, IL-12, and TNF-α) were increased at the early stage (day 3) and decreased at the later stage (day 14). Serum MIP-2 expression level was steadily increased in all three groups from early to late stages, but significantly higher in ΔmexB group than in K767 and nalB groups ( Δ P<0.05). In conclusion, the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection. High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.
Animals ; Bacterial Outer Membrane Proteins ; metabolism ; Lung ; microbiology ; Membrane Transport Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Pseudomonas Infections ; metabolism ; microbiology ; Pseudomonas aeruginosa ; metabolism

OBJECTIVETo determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism.

METHODSOmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays.

RESULTSThe bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01).

CONCLUSIONExpression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Antigens, Bacterial ; genetics ; metabolism ; Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cell Line ; Chaperonin 60 ; genetics ; metabolism ; Humans ; Leptospira interrogans ; genetics ; immunology ; pathogenicity ; Lipoproteins ; genetics ; metabolism ; Macrophages ; microbiology

To study the resistant mechanism and clinical significance of pseudomonas aeruginosa to beta-lactam antibiotics, the outer membrane permeability rate of 30 P. aeruginosa strains to 5 beta-lactam antibiotics was measured and their production of beta-lactamase and the beta-lactamase genes they carried detected. Furthermore, the relationship between the permeability, beta-lactamase and the clinical effects of beta-lactam antibiotics was observed. By using 14C-penicillin and liquid-scintillant isotope assay, the affinity of penicillin binding proteins (PBPS) was measured and their roles in the resistant mechanism studied. It was revealed that the permeability rate was higher in sensitive strains than in resistant ones (P < 0.05). All strains harbored 1-4 beta-lactamase genes and produced beta-lactamase. Higher permeability rate and higher degree of stability to beta-lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the permeability and beta-lactamase. These results suggested that the permeability of outer membrane and beta-lactamase, but not PBPs, played important roles in the resistant mechanism of P. aeruginosa to beta-lactam antibiotics and affected the clinical therapeutic effectiveness of some patients.
Anti-Bacterial Agents/*pharmacology ; Bacterial Outer Membrane Proteins/metabolism ; Microbial Sensitivity Tests ; Permeability ; Pseudomonas aeruginosa/*drug effects ; beta-Lactam Resistance/*genetics ; beta-Lactamases/metabolism ; beta-Lactams/*pharmacology

To study the resistant mechanism and clinical significance of pseudomonas aeruginosa to beta-lactam antibiotics, the outer membrane permeability rate of 30 P. aeruginosa strains to 5 beta-lactam antibiotics was measured and their production of beta-lactamase and the beta-lactamase genes they carried detected. Furthermore, the relationship between the permeability, beta-lactamase and the clinical effects of beta-lactam antibiotics was observed. By using 14C-penicillin and liquid-scintillant isotope assay, the affinity of penicillin binding proteins (PBPS) was measured and their roles in the resistant mechanism studied. It was revealed that the permeability rate was higher in sensitive strains than in resistant ones (P < 0.05). All strains harbored 1-4 beta-lactamase genes and produced beta-lactamase. Higher permeability rate and higher degree of stability to beta-lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the permeability and beta-lactamase. These results suggested that the permeability of outer membrane and beta-lactamase, but not PBPs, played important roles in the resistant mechanism of P. aeruginosa to beta-lactam antibiotics and affected the clinical therapeutic effectiveness of some patients.
Anti-Bacterial Agents ; pharmacology ; Bacterial Outer Membrane Proteins ; metabolism ; Humans ; Microbial Sensitivity Tests ; Permeability ; Pseudomonas aeruginosa ; drug effects ; beta-Lactam Resistance ; genetics ; beta-Lactamases ; metabolism ; beta-Lactams ; pharmacology

OBJECTIVETo construct a fused expression vector of the outer membrane protein gene PorB of Neisseria gonorrhoeae, express the fusion protein in the prokaryotic system, and obtain a gene recombination protein, for the purpose of preparing the ground for further research on the pathopoiesis and immune protective response of PorB.

METHODSA pair of primers were designed according to the known sequence of the PorB gene, and the PorB gene was amplified by PCR from the genome of Neisseria gonorrhoeae 29403 and cloned into the prokaryotic expression plasmid pGEX-4T-1 to generate pGEX-4T-PorB recombinants. The recombinant plasmid pGEX4T-PorB was transferred into competent cells E. coli BL21. After confirmed by restriction endonuclease digestion, PCR and DNA sequencing analysis, the recombinant protein was induced to express by isopropyl-beta-D-thiogalactoside (IPTG), and examined by SDS-PAGE and Western blotting.

RESULTSRestriction endonuclease digestion, PCR amplification and DNA sequencing analysis showed that the PorB gene of 1 047 bp was amplified from Neisseria gonorrhoeae DNA, and the recombinant plasmid pGEX-4T-PorB was successfully constructed and highly expressed in E. coli.

CONCLUSIONThe prokaryotic expression vector of pGEX-4T-PorB was successfully constructed and efficiently expressed in the prokaryotic system, which has provided a basis for further study on the biological activity of the PorB protein, as well as animal immune experiment and detection of Neisseria gonorrhoeae, and its application as a mucosal immune vaccine.

Bacterial Outer Membrane Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; metabolism ; Gene Expression ; Genetic Vectors ; Molecular Sequence Data ; Neisseria gonorrhoeae ; genetics ; metabolism ; Plasmids ; Porins ; genetics ; metabolism

OBJECTIVETo determine the location on outer envelope and natural antibody response and types of genus-specific lipoprotein antigen LipL41s in patients with Leptospira interrogans.

METHODSMicroscope agglutination test (MAT) was used to examine leptospirosis patients' serum samples from Sichuan area, China. Ni-NTA affinity chromatography was performed to extract the target recombinant rLipL41/1 and rLipL41/2 products that expressed under inducement of IPTG. Western blot assay was performed to detect the immunoreactivity between the sera from the patients infected with different serogroups of L. interrogans and rLipL41s. Immune aurosol electron microscopy was selected to locate the position of LipL41s on leptospiral envelope. ELISA based on rLipL41s was established to confirm the level and types of specific antibody.

RESULTSL. interrogans serogroup icterohaemorrhagiae remained to be the most dominant leptospiral serogroup in Sichuan area. All the sera from patients infected with different serogroups of L. interrogans could efficiently recognize the LipL41s which were the protein molecular that located on the external surface of leptospiral envelope. In the 156 serum samples from MAT positive leptospirosis patients, the positive rates for rLipL41/1 or rLipL41/2 specific IgM appeared to be 84.6%-87.8% and 78.2%-83.3%, respectively, while for rLipL41/1 or rLipL41/2 specific IgG they were 69.2%-81.4% and 75.0%-80.1%, respectively.

CONCLUSIONLipL41s were the leptospiral superficial protein antigen of L. interrogans. Both the LipL41/1 and LipL41/2 could induce serum antibodies IgM and IgG with extensive antigenic-cross reaction during natural infection of L. interrogans in general populations. Hence, rLipL41/1 or rLipL41/2 could be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.

Antibodies, Bacterial ; blood ; immunology ; Antigens, Bacterial ; metabolism ; Bacterial Outer Membrane Proteins ; metabolism ; Cell Membrane ; metabolism ; Cross Reactions ; Humans ; Immunoglobulin G ; blood ; immunology ; Immunoglobulin M ; blood ; immunology ; Leptospira interrogans ; metabolism ; ultrastructure ; Leptospirosis ; immunology ; Species Specificity

This study was aimed to construct a prokaryotic expressing vector of Hap gene from Nontypeable Haemophilus influenzae, and express and identify the fusion proteins of Hap-His in E. coli. The gene encoding protein Hap was amplified from Nontypeable Haemophilus influenzae ATCC49247 chromosomal DNA by PCR, then it was cloned into prokaryotic expression plasmid pET-32a (+). The recombinant plasmid pET-32a(+)-Hap was transformed into E. coli BL21 and expression was induced by Isopropy-beta-D-thiogalatoside(IPTG). The Hap-His fusion protein expressed so was analyzed by SDS-PAGE and Western-blot. The results showed that the recombinant expressive plasmid pET-32a (+)-Hap was constructed successfully, and the recombinant plasmid expressed Hap-His fusion protein with relative molecule mass 176 000 and mainly existed in inclusion body. This fusion protein could combine with anti-His monoclonal antibody specifically through Western blot analysis. Successful expression of Hap-His fusion protein in prokaryotic cell could lay a basis for further study of immunocompetence of Hap protein and development of NTHi vaccine.
Bacterial Outer Membrane Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Haemophilus influenzae ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serine Endopeptidases ; biosynthesis ; genetics

OBJECTIVETo construct prokaryotic expression systems of ltB/ctB-ompL1/1 fusion genes and to determine the L.interrogans carrying status in leptospirosis patients with the expression products.

METHODSThe fusion genes ltB-ompL1/1 and ctB-ompL1/1 were constructed using linking primer PCR method. SDS-PAGE was used to examine expression of the target recombinant proteins rLTB-rOmpL1/1 and rCTB-rOmpL1/1. Western blot and GM1-ELISA were used to measure the immunogenic and GM(1)-binding activities of rLTB-rOmpL1/1 and rCTB-rOmpL1/1, respectively. PCR and MAT were performed to detect the expression of ompL1 gene in 97 wild L.interrogans strains. Antibodies against ompL1 gene products in serum samples of 228 leptospirosis patients were detected with ELISA method.

RESULTSThe homogeneity of nucleotide and putative amino acid sequence of ltB-jompL1/1 and ctB-ompL1/1 fusion genes were 99.7 % - 99.9 % and 99.5 % - 100 %, in comparison with the reported corresponding sequences. Expression outputs of both rLTB-rOmpL1/1 and rCTB-rOmpL1/1, mainly present in inclusion body, accounted for 10% of the total bacterial protein. Both rLTB-rOmpL1/1 and rCTB-rOmpL1/1 could combine to rabbit anti-rOmpL1/1 serum and bovine GM(1). 89.7 % of L.interrogans wild strains had ompL1 gene. 87.6% of the wild L.interrogans strains presented positive results for MAT (titers :1:4-1:256) with the rabbit anti-rOmpL1/1 or anti-rOmpL1/2 sera. 86.8% and 88.6% of the patients' serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies, respectively.

CONCLUSIONThe fusion proteins, rLTB-rOmpL1/1 and rCTB-rOmpL1/1, showed high immunogenic and GM(1)-binding activities. ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and its products expressed by different genotypes exhibit extensive cross-antigenicity.

Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; Bacterial Vaccines ; genetics ; Cloning, Molecular ; Enterotoxins ; genetics ; Escherichia coli Proteins ; genetics ; Genes, Bacterial ; genetics ; Humans ; Leptospira interrogans ; genetics ; immunology ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccines, Synthetic ; genetics