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

OBJECTIVE: This study aims to use Arginine-gingipain A gene vaccine (pVAX1-rgpA) to immunize adult Beagle dogs and to evaluate its effect during peri-implantitis progression and development. METHODS: Plasmid pVAX1-rgpA was constructed. The second and third bilateral mandible premolars of 15 adult Beagle dogs were extracted, and the implants were placed immediately. After 3 months, the animals were randomly divided into groups A, B, and C. Afterward, the animals were immunized thrice with plasmid pVAX1-rgpA, with heat-killed Porphyromonas gingivalis, or pVAX1, respectively. IgG in the serum and secretory IgA (sIgA) in saliva were quantitatively analyzed by enzyme-linked immunosorbent assay before and after 2 weeks of immunization. Peri-implantitis was induced with cotton ligatures fixed around the neck of implants. Probing depth (PD) and bleeding on probing were recorded. All animals were sacrificed after ligaturation for 6 weeks. Decalcified sections with thickness of 50 μm were prepared and dyed with methylene blue to observe the bone phenotype around implants. RESULTS: Levels of serum IgG and sIgA in saliva were higher in groups A and B after immunization than before the process (P<0.05) and higher than those in group C (P<0.05). However, no difference was observed between groups A and B (P>0.05). At 4 and 6 weeks after ligaturation, PD of the ligatured side in group C was higher than that in groups A and B (P<0.05). On the other hand, no difference was identified between groups A and B (P>0.05). Bone loss in group A was significantly lower than that of the other groups (P<0.05). Abundant inflammatory cells and bacteria were present in the bone loss area around the implants in the three groups, as identified through hard tissue section observation. However, group C presented the most number of inflammatory cells and bacteria in the bone loss area around the implants. CONCLUSIONS IgG and sIgA can be generated by immunity with rgpA DNA vaccine, which can significantly slow down bone loss during experimental peri-implantitis in dogs.
Adhesins, Bacterial ; therapeutic use ; Alveolar Bone Loss ; Animals ; Arginine ; Cysteine Endopeptidases ; therapeutic use ; Dental Implants ; Dogs ; Peri-Implantitis ; prevention & control ; Porphyromonas gingivalis ; chemistry ; Vaccines ; therapeutic use

OBJECTIVETo investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins.

METHODSSpecific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination.

RESULTSThe target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P>0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P<0.05). The PepO, PsaA, and combined immunization groups had significantly higher protection rates for mice infected with Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P<0.05). The PepO and combined immunization groups had a significantly higher protection rate for mice infected with Streptococcus pneumoniae D39 than the PsaA group (P<0.05). The results of colonization experiment showed that compared with the control group, the PepO, PsaA, and combined immunization groups showed a significant reduction in the colonization of Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P<0.05). The combined immunization group showed a better effect on reducing the colonization of CMCC31207 in the lung than the PepO and PsaA alone groups.

CONCLUSIONSCombined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.

Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; analysis ; Bacterial Proteins ; immunology ; Female ; Immunization ; Lipoproteins ; immunology ; Lung ; microbiology ; Metalloendopeptidases ; immunology ; Mice ; Mice, Inbred BALB C ; Pneumococcal Infections ; prevention & control ; Pneumococcal Vaccines ; immunology ; Saliva ; immunology

Staphylococcus aureus is a major cause of hospital-acquired infection. Because the bacteria are very easy to become resistant to antibiotics, vaccination is a main method against S. aureus infection. Clumping factor B (ClfB) is an adhesion molecule essential for S. aureus to colonize in the host mucosa and is regarded as an important target antigen. In this study, we successfully used Escherichia coli to express a segment encoding the N1-N3 regions of ClfB protein (Truncated-ClfB) cloned from S. aureus. The protein was purified by affinity and ion exchange chromatographies and gel filtration. Rabbits were immunized three times with purified Truncated-ClfB. After that, blood was collected to prepare serum which were then used for measurement of antibody level. Phagocytosis of S. aureus opsonized by the serum was determined by a flow cytometry. Results show that the serum IgG titer reached 1:640 000. Phagocytosed S. aureus by polymorphonuclear leukocytes were significantly more when the bacteria were opsonized by the serum from Truncated-ClfB immunized rabbits than those from no immunized group (P < 0.01). Therefore, the results indicated that Truncated-ClfB could be a promising vaccine candidate against S. aureus infection.
Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; blood ; Escherichia coli ; Flow Cytometry ; Immune Sera ; Immunoglobulin G ; blood ; Opsonin Proteins ; immunology ; Phagocytosis ; Rabbits ; Staphylococcal Infections ; immunology ; Staphylococcus aureus

Adhesins, Bacterial ; Biomedical Research ; Fusobacterium nucleatum

OBJECTIVETo establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak).

METHODSGingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis.

RESULTSArginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1.

CONCLUSIONS1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.

Adhesins, Bacterial ; pharmacology ; Animals ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Bcl-2-Like Protein 11 ; Cell Line ; Cysteine Endopeptidases ; pharmacology ; Humans ; MCF-7 Cells ; Membrane Proteins ; metabolism ; Mice ; Osteoblasts ; cytology ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Tosyllysine Chloromethyl Ketone ; pharmacology ; bcl-2 Homologous Antagonist-Killer Protein ; metabolism ; bcl-2-Associated X Protein ; metabolism

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

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

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