As an important protein structure on the surface of bacteria, type Ⅳ pili (TFP) is the sensing and moving organ of bacteria. It plays a variety of roles in bacterial physiology, cell adhesion, host cell invasion, DNA uptake, protein secretion, biofilm formation, cell movement and electron transmission. With the rapid development of research methods, technical equipment and pili visualization tools, increasing number of studies have revealed various functions of pili in cellular activities, which greatly facilitated the microbial single cell research. This review focuses on the pili visualization method and its application in the functional research of TFP, providing ideas for the research and application of TFP in biology, medicine and ecology.
Fimbriae, Bacterial/metabolism* ; Bacterial Proteins/genetics* ; Bacterial Physiological Phenomena ; Bacterial Adhesion/physiology*

OBJECTIVETo investigate the mechanism of monocyte chemoattractant protein-1 (MCP-1) regulations of human gingival fibroblasts (HGF) by challenge of Porphyromonas gingivalis (Pg) with different fimA genotypes.

METHODSPg ATCC 33277 (type I), WCSP115 (type II), WCSP1.5 (type III), W83 (type IV) were assessed for their inductions of MCP-1 expression in HGF. MCP-1 mRNA levels of HGF were determined by real-time RT-PCR and MCP-1 protein levels in culture supernatant by ELISA at different time intervals (1 h, 3h, 6h and 12h) following continuous co-culture of bacteria with HGF.

RESULTSMCP-1 mRNA and protein levels were both up-regulated when HGF co-cultured with different Pg fimA genotypes. Type II was stronger than other fimA genotypes, HGF expressed significantly great amount of MCP-1 mRNA [(25.75 +/- 3.12)-(326.69 +/- 35.35)] and protein [(178.20 +/- 46.20)-(443.46 82.19) ng/L] for different time periods; While Type III was weaker than other fimA genotypes, and the level of MCP-1 mRNA was [ (4.16 +/- 0.82)-(94.17 +/- 18.56)] and protein [(86.95 +/- 23.90)-(264.01 +/- 28.59) ng/L](P < 0.05).

CONCLUSIONSfimA genotypes of Pg are related with the inductions of MCP-1, which might indicate fimA genotype is associated with pathogenesis of Pg.

Cells, Cultured ; Chemokine CCL2 ; metabolism ; Fibroblasts ; metabolism ; Fimbriae Proteins ; genetics ; Genotype ; Gingiva ; cytology ; microbiology ; Humans ; Porphyromonas gingivalis ; genetics

K88ac-LT(B) gene derived from pQE30-K88ac-LT(B) was cloned into the expression vector pLA and then the recombinant vector was transformed into the competent cells Lactobacillus casei 525. The recombinant bacteria were grown at 37 degrees C, in MRS broth. Western blotting analysis with rabbit-anti-K88ac-LT(B) polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 71.2 kD. The K88ac-LT(B) fusion protein on the cell surface was confirmed by immunofluorescence mciroscopy and flow cytometric analysis. In addition, the survival of recombinant Lactobacillus casei 525 was studied in imitative gastrointestinal environments such as artificial gastro fluid (pH 1.5-5.5), artificial intestinal fluid, bile(0.3-3.0 g/L). The results indicated that the recombinant strain survived well in artificial gastric fluids at pH 2.5-4.5 in 5 h. The recombinant Lactobacillus casei 525 could slowly grow in the artificial intestinal fluid for different time, and could survive in 0.3% bile.
Antigens, Bacterial ; genetics ; Bacterial Toxins ; genetics ; metabolism ; Enterotoxigenic Escherichia coli ; genetics ; metabolism ; Enterotoxins ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; Fimbriae Proteins ; genetics ; Gastric Juice ; Lactobacillus casei ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombination, Genetic

OBJECTIVETo investigate the mechanism of matrix metalloproteinases (MMP) regulations of human gingival fibroblasts (HGF) by challenge of Porphyromonas gingivalis (Pg) with different fimA genotypes.

METHODSPg ATCC 33277 (type I), WCSP115 (type II), WCSP1.5 (type III), W83 (type IV) were assessed for their inductions of MMP-1 and MMP-2 expression in HGF. MMP mRNA levels of HGF were determined by real-time RT-PCR and MMP protein levels in culture supernatant were determined by ELISA at different time intervals (1, 3, 6 and 12 h) following continuous co-culture of bacteria with HGF.

RESULTSWhen co-cultured with Pg, the MMP-1 and MMP-2 mRNA and protein expression of HGF significantly increased compared with the negative control group (P < 0.01). The group of type II showed greater up-regulated than other fimA genotypes in the mRNA and protein expressions of MMP-1 and MMP-2, MMP-1 mRNA [(28.88 +/- 3.12) - (231.01 +/- 24.99)] and protein [(1.35 +/- 0.17) - (3.08 +/- 1.20)] microg/L; MMP-2 mRNA [(20.42 +/- 2.21) - (188.34 +/- 37.37)] and protein [(2.57 +/- 0.76) - (18.08 +/- 1.15)] microg/L for different time periods; While the group of type III was weaker than other fimA genotypes, the level of MMP-1 mRNA was [(5.11 +/- 0.55) - (72.84 +/- 8.84)] and protein [(0.68 +/- 0.13) - (1.46 +/- 0.94)] microg/L, MMP-2 mRNA [(4.55 +/- 0.55) - (25.75 +/- 3.12)] and protein [(2.28 +/- 0.93) - (11.22 +/- 2.46)] microg/L (P < 0.05).

CONCLUSIONSPg could induce HGF to over-express MMP, and fimA genotypes of Pg may be related to this pathogenicity, which might indicate fimA genotype is associated with pathogenesis of Pg.

Cells, Cultured ; Coculture Techniques ; Fibroblasts ; metabolism ; Fimbriae Proteins ; genetics ; Genotype ; Gingiva ; cytology ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Porphyromonas gingivalis ; genetics ; RNA, Messenger ; genetics

In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; immunology ; metabolism ; Escherichia coli Proteins ; genetics ; immunology ; metabolism ; Escherichia coli Vaccines ; immunology ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; Mice ; Porins ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo construct a recombinant plasmid containing Lgeionella pneumophila pilE gene, detect its expression in NIH3T3 cells and evaluate its immunogenicity.

METHODSPilE gene (LP) was amplified from Legionella pneumophila DNA by PCR and inserted into pcDNA3.1(+) vector to construct the recombinant plasmid pcDNA3.1-pilE, which as verified by restriction endonuclease digestion, PCR and DNA sequencing analysis. NIH3T3 cells were transfected with the recombinant plasmid with Lipofection strategy. Transient and stable pilE gene products were detected by immunofluorescence and Western blotting, respectively. To evaluate the immunogenicity of pcDNA3.1-pilE, the recombinant plasmid was used as a DNA vaccine to immunize female BALB/c mice intramuscularly and the specific antibodies, lymphocyte proliferation response, interferon (IFN)-gamma production and cytotoxic T-lymphocyte response of the immunized mice were detected and evaluated.

RESULTSThe pilE gene of 429 bp in length was amplified. After transfection of NIH3T3 cells with the recombinant plasmid, strong green fluorescence was observed on the cell membrane and inside the cell. A protein with relative molecular mass of 15.7 kD was detected in the transfected cells with Western blotting, suggesting successful protein expression of pilE gene. pcDNA3.1-pilE resulted in much stronger immune response in the immunized mice than pcDNA3.1(+) (P<0.01).

CONCLUSIONThe recombinant plasmid containing Lgeionella pneumophila pilE gene constructed in this study is capable of expression in NIH3T3 cells, and can induce specific humoral and cellular immune responses in mice.

Animals ; Blotting, Western ; Cell Proliferation ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Fimbriae, Bacterial ; Fluorescent Antibody Technique ; Humans ; Immunization ; methods ; Injections, Intramuscular ; Interferon-gamma ; metabolism ; Legionella pneumophila ; genetics ; immunology ; metabolism ; Lymphocytes ; cytology ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; NIH 3T3 Cells ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediates adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by Ligand blot, protein bands with a set of 32-35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC(RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequences of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The above results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins.
Adhesins, Escherichia coli ; metabolism ; Amino Acid Sequence ; Animals ; Enterotoxigenic Escherichia coli ; metabolism ; pathogenicity ; Escherichia coli Infections ; microbiology ; veterinary ; Fimbriae Proteins ; metabolism ; Fimbriae, Bacterial ; chemistry ; Histones ; genetics ; metabolism ; Host-Pathogen Interactions ; Intestinal Mucosa ; metabolism ; Molecular Sequence Data ; Receptors, Cell Surface ; genetics ; metabolism ; Swine

Sodium houttuyfonate (SH) is a derivative of effective component of a Chinese material medica, Houttuynia cordata, which is applied in anti-infection of microorganism. But, the antimicrobial mechanisms of SH still remain unclear. Here, we firstly discovered that SH effectively inhibits the three types of virulence related motility of.Pseudomonas aeruginosa, i.e., swimming, twitching and swarming. The plate assay results showed that the inhibitory action of SH against swimming and twitching in 24 h and swarming in 48 h is dose-dependent; and bacteria nearly lost all of the motile activities under the concentration of 1 x minimum inhibitory concentration (MIC) (512 mg x L(-1) same as azithromycin positive group (1 x MIC, 16 mg x L(-1)). Furthermore, we found that the expression of structural gene flgB and pilG is down-regulated by SH, which implies that inhibitory mechanism of SH against motility of P. aeruginosa may be due to the inhibition of flagella and pili bioformation of P. aeruginosa by SR Therefore, our presented results firstly demonstrate that SH effectively inhibits the motility activities of P. aeruginosa, and suggest that SH could be a promising antipseudomonas agents in clinic.
Alkanes ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Biofilms ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Fimbriae, Bacterial ; drug effects ; genetics ; metabolism ; Houttuynia ; chemistry ; Pseudomonas aeruginosa ; cytology ; drug effects ; genetics ; pathogenicity ; Sulfites ; pharmacology ; Virulence ; drug effects

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.
Administration, Oral ; Agrobacterium tumefaciens ; Animals ; Antigens, Bacterial/genetics/metabolism ; Bacterial Vaccines/administration & dosage/adverse effects/*pharmacology ; Edema Disease of Swine/*immunology/microbiology ; Escherichia coli Infections/immunology/microbiology/*veterinary ; Escherichia coli Proteins/*genetics/metabolism ; Female ; Fimbriae Proteins/genetics/metabolism ; Genetic Engineering ; Intestines/immunology/microbiology/pathology ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Plants, Genetically Modified/*genetics/metabolism ; Seeds/genetics/metabolism ; Shiga Toxin 2/genetics/metabolism ; Shiga-Toxigenic Escherichia coli/genetics/immunology/*pathogenicity ; Swine ; Tobacco/*genetics/metabolism ; Virulence Factors/genetics/metabolism

A pair primer was designed by Oligo 6.0 according to the pilA gene sequence of E. coli isolated from human in GenBank. The pilA Gene was obtained by PCR with the enteropathogenic E. coli isolated from ducks as template and cloned into pMD18-T vector. It was identified by PCR, restriction endonuclease analysis, DNA sequencing and then subcloned into BamH I/Hind III site of prokaryotic expression vector pET-32a(+) and recombinant expression plasmid pET-32a-pilA was constructed successfully. The plasmid was transformed into Eschericha coli BL21 (DE3) and 36kD pilA recombinant protein was expressed be induced with IPTG. The protein was purified by Ni-agarose affinity chromatograghy and was prepared as vaccine with Freund' s adjuvant. The ducklings were immunized with the vaccine at 1 and 8-day-old respectively. Two weeks after last immunized, the antibody titer of duck serum was detected by ELISA and the ducklings were challenged with 10(9) PFU enteropathogenic E. coli GH1.2 virulent strain. The immunoprotection effect of pilA recombinant protein vaccine was evaluated according to the mortality, re-isolated rate of E. coli, and grades of pathological changes. The results show that the antibody titer are 1:12800, but 1:200 were detected from ducklings immunized with homologous whole cells E. coli inactivated vaccine. The mortality, re-isolated rate of E. coli, degree of pathological changes of immunized ducklings is lower than that of the control ducklings and showed significant or extremely significant differences (P < 0.01 or P < 0.05), but non-significant difference compared to the ducklings which immunized with homologous whole cells E. coli inactivated vaccine (P > 0.05). The results show that pilA recombinant protein has some immunoprotection effect with the challenging of virulent strains of E. coli GH1.2.
Animals ; Animals, Newborn ; Antibodies, Bacterial ; blood ; immunology ; Ducks ; microbiology ; Electrophoresis, Polyacrylamide Gel ; Enteropathogenic Escherichia coli ; genetics ; immunology ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli Proteins ; genetics ; immunology ; metabolism ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Humans ; Immunization ; Polymerase Chain Reaction ; Poultry Diseases ; immunology ; mortality ; prevention & control ; Recombinant Proteins ; administration & dosage ; immunology ; metabolism ; Survival Rate ; Virulence