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*

Animals ; Antibodies, Bacterial ; blood ; Cholera Toxin ; immunology ; Cholera Vaccines ; immunology ; Fimbriae, Bacterial ; immunology ; Mice ; Vibrio cholerae O139 ; immunology

OBJECTIVETo construct the recombinant plasmid pPHU281_A_Spec_B, which knock out Porphyrmonas gingivalis (Pg) FimA gene.

METHODSGenomic DNA was extracted from PgATCC33277 which was cultured in anaerobic condition. The upstream and downstream gene of FimA was cloned from Pg genenomic DNA with specific restriction sites by polymerase chain reaction. Suicide vector pPHU281 was inserted by three fragments: upstream, downstream of FimA gene and spectinomycin resistance gene. The recombinant plasmid was confirmed by electrophoresis and sequenced after amplification in compentent cells DH-5α.

RESULTSThe gene sequence was identified by DNA sequencing analysis. The recombinant plasmid pPHU281_A_Spec_B was successfully constructed.

CONCLUSIONSThe recombinant plasmid pPHU281_A_Spec_B was constructed, which may be used for the constructon of FimA deficient Pg.

Base Sequence ; DNA, Bacterial ; genetics ; Fimbriae Proteins ; genetics ; Gene Knockout Techniques ; Genes, Bacterial ; Genetic Vectors ; Plasmids ; genetics ; Porphyromonas gingivalis ; genetics ; Recombinant Proteins ; genetics ; Sequence Analysis, DNA

Adhesins, Escherichia coli ; genetics ; Bacterial Typing Techniques ; Escherichia coli Proteins ; genetics ; Fimbriae Proteins ; genetics ; Genes, Bacterial ; Genotype ; Uropathogenic Escherichia coli ; classification ; genetics

To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25+ Treg cells are stimulated, but for arthritis CD39+ Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-beta and interleukin-10.
Animals ; Antigens, Bacterial/*immunology ; Arthritis, Rheumatoid/immunology/prevention & control ; Autoantigens/*immunology ; Fimbriae Proteins/*immunology ; Humans ; Multiple Sclerosis/immunology/prevention & control ; Salmonella/*immunology ; T-Lymphocytes, Regulatory/*immunology ; *Vaccination

Escherichia coli (E. coli) has ability to express thin aggregative fimbriae, known as curli, on the cell surface. Previously, a few example of curli expression in serogroup O157:H7 of enterohemorrhagic E. coli (EHEC) were reported, compared to other E. coli groups. However, significance of curliation in the EHEC pathobiology has not been described well in the literature. A highly curliated O157:H7 strain was used in this study in order to elucidate role of curliation in EHEC adherence to cultured HEp-2 cells. The expression of curli in the EHEC isolate was consistent with strong positive indication of Congo-red (CR) binding and formation of clumps in the bottom of the tube containing Luria-Bertani (LB) broth when cultured overnight at 37 degress C. A few CR-binding negative (CR-) colonies occurred spontaneously within the population of CR+ isolate. The CR+ EHEC showed massive aggregative adhesion pattern, whereas the spontaneous CR- strain showed typical localized adherence on HEp-2 cells. Electron microscopy confirmed highly curliated bacteria in the CR+ EHEC sample. Interestingly, the curliation disappeared in a msbB1 and msbB2 double mutant derived from the CR+ EHEC. These results suggest that the compromised outer membrane integrity caused by msbB mutations may abrogate curli production in the CR+ EHEC harbouring penta-acylated lipid A structure in their outer membrane.
Bacterial Adhesion/*physiology ; Bacterial Outer Membrane Proteins/*physiology ; Cell Aggregation ; Cells, Cultured ; Epithelial Cells/*microbiology ; Escherichia coli O157/pathogenicity/*physiology/ultrastructure ; Fimbriae, Bacterial/*metabolism/ultrastructure ; Humans ; Larynx/cytology/microbiology ; Microscopy, Electron

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

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

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

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