OBJECTIVETo investigate the distribution of babA2 cagA and vacA genotypes of Helicobacter pylori (Hp) in chronic gastritis and peptic ulcer and to discuss the relationship between babA2, cagA and vacA genotypes of Hp and chronic gastritis and peptic ulcer.

METHODSbabA2, cagA genotypes and vacA subtype of 58 Hp strains isolated from patients of Zhejiang province with chronic gastritis or peptic ulcer were tested by polymerase chain reaction.

RESULTSThe positive rates of babA2, cagA, vacAs1a, vacA m1 and vacA m2 of 58 Hp strains were 87.9%, 100%, 93.1%, 1.7% and 65.5%, respectively. There were no significant differences between the positive rates of babA2, vacA s1a and vacA m2 genes of Hp strains isolated from patients with chronic gastritis and peptic ulcer.

CONCLUSIONThe genotypes of Hp isolated from patients of Zhejiang province were predominatly babA2 positive, cagA positive and vacA s1a/m2. The relationships between babA2, cagA and vacA genotypes of Hp and chronic gastritis and peptic ulcer can not be identified.

Adhesins, Bacterial ; Antigens, Bacterial ; Bacterial Proteins ; genetics ; Carrier Proteins ; genetics ; Chronic Disease ; Gastritis ; microbiology ; Genotype ; Helicobacter pylori ; genetics ; Humans ; Peptic Ulcer ; microbiology

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

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

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD(50) of 4.0 x 10(5) CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD(50) of 1.2 x 10(12) CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes.
Adhesins, Escherichia coli/*genetics/*metabolism ; Animals ; Chickens ; Escherichia coli/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Plasmids/*genetics/metabolism ; Poultry Diseases/*microbiology ; Sepsis/*microbiology

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

OBJECTIVETo detect and compare the activity and intensity of gingipain K (Kgp)-caspase like subdomain in culture medium and cell extract of Porphyromonas gingivalis (Pg) isolates in puberty gingivitis and to reveal the possible association of Kgp with puberty gingivitis.

METHODSThirty-six children of 14 to 17 years old were enrolled in this study. Clinical parameters including gingival index (GI), sulcus bleeding index (SBI) and probing depth (PD) were evaluated. Subgingival plaque samples were collected and Pg isolates were obtained. 16S rRNA PCR was used to confirm Pg clinical isolates. Bacteria were grown in batches of BHI base and harvested at the end of log-phase growth. Culture fractions (culture medium and cell extract) of 10 Pg isolates were performed with SDS-PAGE and Western blot technique using primary antibody against specific Kgp-caspase like subdomain. Activity of Kgp in both samples was detected as well. The data were statistically analyzed using SPSS 11.5 software. The relationship between the Kgp intensity/activity of Kgp and the clinical parameters was statistically analyzed using Spearman correlation coefficient.

RESULTSThere was positive correlation between the intensity/activity of Kgp and the clinical parameters (P < 0.05).

CONCLUSIONSThe Kgp in clinical isolates of Pg from puberty gingivitis is in complicated forms. Caspase-like molecules with low molecular weight may exist as intracellular functional protein molecules which can affect the interaction between Pg and host. Kgp was contributes in certain degree to the pathogenesis of puberty gingivitis.

Adhesins, Bacterial ; genetics ; metabolism ; Adolescent ; Cysteine Endopeptidases ; genetics ; metabolism ; Dental Plaque ; microbiology ; Female ; Gingivitis ; enzymology ; microbiology ; Humans ; Male ; Porphyromonas gingivalis ; genetics ; isolation & purification ; metabolism

OBJECTIVETo analyse the genetic polymorphism of Arg-gingipainB (rgpB), a virulent factors of Porphyromonas gingivalis (P.gingivalis) and discuss the role of the different genotypes in the genesis and progress of chronic periodontitis.

METHODSA total of 104 subgingival plaque samples were included in this study. The extracted DNA was amplified with the primers designed to obtain the gene encoding the catalytic domain of rgpB (rgpB-cd), P.gingivalis was typed into four genotypes by restriction fragment length polymorphism (RFLP).

RESULTSIn lesion site, the detection rate of type IV was the highest (52.78%), which was higher than those of type I and III (P < 0.05 or P < 0.01). While in non-lesion site, The detection rate of type II was the highest (75.86%), which was higher than those of other types (P < 0.01).

CONCLUSIONSThe polymorphism of rgpB-cd gene may influence the virulence of P.gingivalis. P.gingivalis type IV may be well related to periodontitis, while type II may be a indigenous flora.

Adhesins, Bacterial ; genetics ; Adult ; Aged ; Chronic Periodontitis ; microbiology ; Cysteine Endopeptidases ; genetics ; Dental Plaque ; microbiology ; Female ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Porphyromonas gingivalis ; genetics ; pathogenicity

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

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