Objective To explore the aptamer specific binding blood group antigen-binding adhesin (BabA) of Helicobacter pylori (H.pylori) for blocking of H.pylori adhering host cell. Methods H.pylori strain was cultured and its genome was extracted as templates to amplify the BabA gene by PCR with designed primers. The BabA gene obtained was cloned and constructed into prokaryotic expression plasmid, which was induced by isopropyl beta-D-galactoside (IPTG) and purified as target. The single stranded DNA (ssDNA) aptamers that specifically bind to BabA were screened by SELEX. Enzyme-linked oligonucleotide assay (ELONA) was used to detect and evaluate the characteristics of candidate aptamers. The blocking effect of ssDNA aptamers on H.pylori adhesion was subsequently verified by flow cytometry and colony counting at the cell level in vitro and in mouse model of infection, respectively. Meanwhile, the levels of cytokines, interleukin 6 (IL-6), IL-8, tumor necrosis factor α (TNF-α), IL-10 and IL-4 in the homogenate of mouse gastric mucosa cells were detected by ELISA. Results The genome of H.pylori ATCC 43504 strains was extracted and the recombinant plasmid pET32a-BabA was constructed. After induction and purification, the relative molecular mass (M_r) of the recombinant BabA protein was about 39 000. The amino acid sequence of recombinent protein was consistent with BabA protein by peptide mass fingerprint (PMF). Five candidate aptamers were selected to bind to the above recombinent BabA protein by SELEX. The aptamers A10, A30 and A42 identified the same site, while A3, A16 and the above three aptamers identified different sites respectively. The aptamer significantly blocked the adhesion of H.pylori in vitro. Animal model experiments showed that the aptamers can block the colonization of H.pylori in gastric mucosa by intragastric injection and reduce the inflammatory response. The levels of IL-4, IL-6, IL-8 and TNF-α in gastric mucosal homogenates in the model group with aptamer treatment were lower than that of model group without treatment. Conclusion Aptamers can reduce the colonization of H.pylori in gastric mucosa via binding BabA to block the adhesion between H.pylori and gastric mucosal epithelial cells.
Animals ; Mice ; Helicobacter pylori/genetics* ; Interleukin-4 ; Interleukin-6 ; Interleukin-8 ; Tumor Necrosis Factor-alpha ; Stomach ; Oligonucleotides ; Adhesins, Bacterial/genetics* ; Blood Group Antigens

Fibronectin-binding protein (FnBPA) is a protein that expresses on cell surface of Staphylococcus aureus during early stage of infection. FnBPA was capable of promoting Staphylococcus aureus to invade cells and was viewed as a potential immune target. Based on the FnBPA-A gene two recombinant expression vectors with or without Kozak sequence were constructed. After identified and confirmed by restriction enzyme digestion and sequencing they were used to immunize C57BL/6 mice. Then induced antibody titer, T lymphocyte proliferative response and experiment mice challenge test were measured. Our result indicates that humoral immune responses and challenge experiment induced by recombinant DNA with Kozak sequence were better than those without Kozak sequence (P < 0.05). For T lymphocyte proliferative response the induced effect of recombinant DNA with Kozak sequence was higher than that without Kozak sequence, but there was no significant difference (P > 0.05). We conclude that Kozak sequence could play an important role in immune response induced by FnBPA-A recombinant DNA.
Adhesins, Bacterial ; genetics ; immunology ; Animals ; Immunization ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins ; immunology ; Staphylococcus aureus ; immunology ; T-Lymphocytes ; immunology ; Vaccines, DNA ; genetics ; immunology

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and anti-inflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4+/+ BMDCs was not observed in TLR4-/- BMDCs. Furthermore, FAP induced DC-mediated CD8+ T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.
*Adhesins, Bacterial/genetics/metabolism ; Animals ; CD8-Positive T-Lymphocytes/metabolism ; *Cancer Vaccines/therapeutic use ; Cell Proliferation ; Cytokines/metabolism ; Dendritic Cells/*cytology ; Disease Models, Animal ; Gene Expression Regulation ; Glycogen Synthase Kinase 3/metabolism ; Humans ; Mice ; Mice, Inbred C57BL ; Mycobacterium avium/genetics/metabolism ; Paratuberculosis/metabolism ; Protein Binding ; Signal Transduction ; T-Lymphocytes, Cytotoxic/metabolism ; *Thymoma/genetics/metabolism ; *Toll-Like Receptor 4/agonists/genetics/metabolism

Bacterial biofilms can be viewed as a specific type of persistent bacterial infection. After initial invasion, microbes can attach to living and non-living surfaces, such as prosthetics and indwelling medical devices, and form a biofilm composed of extracellular polysaccharides, proteins, and other components. In hosts, biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. The clinical aspects of biofilm formation and leading strategies for biofilm inhibitors will be discussed in this mini-review.
Adhesins, Bacterial ; drug effects ; physiology ; Aminoacyltransferases ; antagonists & inhibitors ; genetics ; Animals ; Antimicrobial Cationic Peptides ; genetics ; pharmacology ; Bacterial Infections ; microbiology ; surgery ; Bacterial Proteins ; antagonists & inhibitors ; genetics ; Biofilms ; drug effects ; growth & development ; Chronic Disease ; Cysteine Endopeptidases ; genetics ; Cysteine Proteinase Inhibitors ; pharmacology ; Humans ; Inflammation ; microbiology ; Quorum Sensing ; drug effects ; physiology ; Wound Infection ; microbiology ; surgery

OBJECTIVEThe P1 protein of Mycoplasma pneumoniae (MP) plays an important role in the pathogenesis of MP pneumonia. It mediates the attachment of the pathogen to host cells and elicits a strong humoral immune response during infection. In early studies, only two types of MP P1 genes were assumed to exist. Later, eight subtypes of MP P1 genes and some variations of P1 gene were reported. However, there are no related reports in China until now. This study aimed to understand epidemiology of MP subtype in Zhejiang province, China, as well as the relationship between MP subtype and clinical severity of MP pneumonia.

METHODClinical samples were collected by nasopharyngeal aspiration from children with MP pneumonia hospitalized in the Children's Hospital of Zhejiang University School of Medicine from February to December in 2009. P1 gene fragment was amplified by using PCR method (with primers of ADH1/ADH2 and ADH3/ADH4, respectively). Then ADH1/ADH2-generated fragments were digested with HaeIII, HpaII, Sau3A, and the ADH3/ADH4-generated fragments digested with HaeIII, Sau3A, HhaI, RsaI. The MP P1 subtypes were determined based on resulting fragments. Part of samples were selected for sequencing. The clinical data of different MP subtype pneumonia were compared.

RESULTA total of 300 hospitalized children with MP pneumonia were enrolled in this study. All the samples produced specific bands for MP P1 gene after PCR with primers of ADH1/ADH2 and ADH3/ADH4 respectively. By restrictive fragment length polymorphism analysis, 297 clinical specimens showed the characteristic band patterns for P1 type 1 identical to Mp129, and only 3 clinical specimens showed the characteristic band pattern for P1 type 2 identical to MP-FH. All P1 type 1 and P1 type 2 showed the same subtype bands respectively, as subtype 1b and 2a. After sequencing, one synonymous point mutation in P1 type 1 was identified relative to the MP129 P1 sequence at nucleotide position (nt) 208(G→A). Three cases with P1 type 2 MP pneumonia were found to have liver damage, and longer hospital stay and fever duration than P1 type 1, but no statistically significant difference was found.

CONCLUSIONClinical samples can be used directly for genotyping of MP. The dominating type of MP in Zhejiang Province was P1 type 1 subtype 1b. But whether there was any relationship between MP subtype and clinical severity remains to be clarified.

Adhesins, Bacterial ; genetics ; Child ; China ; DNA, Bacterial ; genetics ; Genotype ; Humans ; Mycoplasma pneumoniae ; genetics ; isolation & purification ; Nasopharynx ; microbiology ; Pneumonia, Mycoplasma ; microbiology ; Polymorphism, Restriction Fragment Length

OBJECTIVETo construct a recombinant Lactobacillus acidophilus that expresses high levels of Helicobacter pylori (Hp) adhesin Hp0410.

METHODSThe gene fragment encoding Hp0410 was amplified by PCR from the DNA of H. pylori NCTC11639 strain and cloned into the shuttle plasmid pMG36e to construct pMG36e-Hp0410, which was transformed into Lactobacillus acidophilus by electroporation. The target protein was confirmed with SDS-PAGE and silver nitrate staining and analyzed by Western blotting. The stability of the recombinant plasmid was assessed by drawing the growth curve of the recombinant Lactobacillus acidophilus.

RESULTSA 750-bp fragment was inserted into the pMG36e plasmid and transformed into Lactobacillus lactis. The transformed bacterium expressed the target protein with a relative molecular mass of about 34 kD. Western blotting confirmed that the expressed proteins could be recognized by the serum of patients with Hp infection. The recombinant plasmid pMG36e-Hp0410 exhibited good stability in the presence or absence of erythromycin.

CONCLUSIONSThe recombinant Lactobacillus acidophilus with high constitutive expression of Hp0410 has been constructed successfully.

Adhesins, Bacterial ; biosynthesis ; genetics ; immunology ; Bacterial Vaccines ; biosynthesis ; Helicobacter Infections ; prevention & control ; Humans ; Lactobacillus acidophilus ; genetics ; metabolism ; Plasmids ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, Attenuated ; biosynthesis

OBJECTIVETo obtain highly purified intimin encoded by the eae gene and study its adhesion activity.

METHODSThe eae gene was amplified from enterohemorrhagic Escherichia coli O157:H7 (EHEC) chromosome by PCR and cloned into pMD19-T vector. The eae gene was cut from pMD19-T vector and subcloned into the prokaryotic expression plasmid pET28a(+), and expressed in E.coli BL21(DE3). The recombinant protein was purified with Ni(2+)-chelating affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purified intimin was detected by immunofluorescence staining to test its adhesion.

RESULTSThe 2805-bp eae gene fragment was obtained, and the recombinant expression plasmid pET28a(+)-eae was successfully expressed in E.coli BL21 (DE3). The molecular weight of the recombinant protein was 97 000. Purified recombinant intimin was recognized by rabbit anti-O157 antiserum, and bound to the surface of HEp-2 cells as revealed by immunofluorescence staining.

CONCLUSIONHighly purified and immunoreactive intimin has been successfully obtained, which can adhere to the surface of HEp-2 cells. The acquisition of recombinant intimin provides the basis for studying its interaction with the host receptors during EHEC O157:H7 infection.

Adhesins, Bacterial ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Animals ; Blotting, Western ; Cell Adhesion ; Cell Line ; Cloning, Molecular ; Escherichia coli ; genetics ; Escherichia coli O157 ; Escherichia coli Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Gene Expression ; Plasmids ; genetics

The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein.
Adhesins, Bacterial ; biosynthesis ; genetics ; immunology ; Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Pasteurella multocida ; chemistry ; Rabbits ; microbiology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Iro system and temperature-sensitive hemagglutinin (Tsh) genes were identified by suppression subtractive hybridization (SSH) and selective capture of transcribed sequences (SCOTS). To get more insights in the distribution and the occurrence of the iroC and tsh genes, we examined 243 avian E. coli strains for the presences of the these genes. Among 243 avian E. coli isolates, iroC gene was present in 84.4% strains (205/243). Of the 205 iroC-positive isolates, iroC gene was found in 184 (89.8%), 18(8.8%) and 3 (1.5%) isolates with high, intermediate and low pathogenicity, respectively. Of the 167 tsh-positive isolates, tsh gene was detected in 146 (87.4%), 21 (12.6%) and 0 (0%) isolates with high, intermediate and low pathogenicity, respectively. Among tsh-positive isolates, 89.5 to 100% of the highly pathogenic isolates of O1, O2 or O78 serogroups had the tsh gene, while 53.3% of the highly pathogenic isolates of non-O1, O2 and O78 serogroups had the tsh gene (P<0.01). Suicide vectors for deletion of the iroBCDEN or tsh genes were constructed as follows. The 715-bp fragments of iroB and 603-bp fragment of the iroN were generated by PCR respectively. Both of these two fragments together with EGFP gene were cloned into pUC18, termed pUC18-iroBNEGFP. A resultant suicide vector containing the iroB-EGFP-iroN fragment was obtained and named pMEG375-iroBNEGFP. Similarly, both of the 685-bp fragment of tshF and the 692-bp fragment of the tshR together with gentamycin gene were cloned into pUC18, resulting in pUC18-tshFRGm. A resultant suicide vector containing the tshF-Gm-tshR fragment was named pMEG375-tshFRGm. Mutant derivatives of strain E037 were generated by allelic replacements and were named E037(Deltairo), E037(Deltatsh) and E037(DeltairoDeltatsh). The 50% lethal dose (LD50) of E037, E037(Deltairo), E037(Deltatsh) and E037(DeltairoDeltatsh) in commercial day-old chickens experimentally inoculated via intratrachea were determined to be 10(5.6), 10(8.4), 10(9.0) and 10(9.5)CFU, respectively. In the chicken challenging model, the mutants were tested to determine the individual role of this system for virulence and persistence in chickens. The result suggested that Iro system and Tsh were important in the pathogenicity of APEC.
Adhesins, Escherichia coli ; genetics ; Animals ; Chickens ; Escherichia coli ; genetics ; pathogenicity ; Escherichia coli Infections ; microbiology ; veterinary ; Genes, Bacterial ; genetics ; Mutation ; Nucleic Acid Hybridization ; methods ; Organisms, Genetically Modified ; Poultry Diseases ; microbiology ; Transformation, Genetic ; Virulence Factors ; genetics

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