hpaA Gene Research Articles

Mechanism of transcription regulation by Acinetobacter baumannii HpaR in the catabolism of p-hydroxyphenylacetate.

HpaR is a transcription regulator in the MarR family that controls the expression of the gene cluster responsible for conversion of p-hydroxyphenylacetate to pyruvate and succinate for cellular metabolism. Here, we report the biochemical and structural characterization of Acinetobacter baumannii HpaR (AbHpaR) and its complex with cognate DNA. Our study revealed that AbHpaR binds upstream of the divergently transcribed hpaA gene and the meta-cleavage operon, as well as the hpaR gene, thereby repressing their transcription by blocking access of RNA polymerase. Structural analysis of AbHpaR-DNA complex revealed that the DNA binding specificity can be achieved via a combination of both direct and indirect DNA sequence readouts. DNA binding of AbHpaR is weakened by 3,4-dihydroxyphenylacetate (DHPA), which is the substrate of the meta-cleavage reactions; this likely leads to expression of the target genes. Based on our findings, we propose a model for how A. baumannii controls transcription of HPA-metabolizing genes, which highlights the independence of global catabolite repression and could be beneficial for metabolic engineering toward bioremediation applications. DATABASES: The structural data that support these findings are openly available in the wwPDB at https://doi.org/10.2210/pdb7EL2/pdb and https://doi.org/10.2210/pdb7EL3/pdb for AbHpaR and AbHpaR-DNA complex, respectively.

Cloning, expression and purification flagellar sheath adhesion of Helicobacter pylori in Escherichia coli host as a vaccination target.

PurposeHelicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori–specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid chromatography (FPLC) as a vaccination target.Materials and MethodsThe hpaA gene was inserted into pET28a (+) as cloning and expression vectors respectively. The recombinant plasmid (pET-hpaA) was subjected to sequencing other than polymerase chain reaction (PCR) and digestion analysis. Protein expression was induced by adding 1 mM isopropyl-β-D-thiogalactoside to cultures of E. coli strain BL21 transformed with pET-hpaA. Protein expression assessed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein purification of flagellar sheath adhesion was by FPLC.ResultsThe restriction endonuclease digestion, PCR amplification analysis showed that the hpaA gene of 730 bp was amplified from H. pylori DNA and sequencing analysis of the pET-hpaA confirmed the cloning accuracy and in frame insertion of hpaA fragment. SDS-PAGE analysis showed the expression of an approximately 29,000 Da protein.ConclusionSequencing results along with SDS-PAGE analysis confirms the expression of recombinant hpaA in the heterologous E. coli BL21. Conclusion A prokaryotic expression system for hpaA gene was successfully constructed. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.

Phage display and immunological identification of efficient T- and B-combined antigenic epitopes in Helicobacter pylori adhesin A

Objective To analyze and determine the efficient T- and B-combined (T/B) antigenic epitopes in Helicobacter pylori adhesin A. Methods Recombinant HpaA (rHpaA) was expressed for immunizing rabbit to generate antiserum. T- and B-cell epitopes in HpaA molecule were predicted by using bioinformatic technique. The segments to encode T/B combined epitope peptides were amplified by PCR and the phage display systems of T/B combined epitopes were subsequently constructed. PEG/NaCl precipitation method was applied to extract the recombinant phage PⅢ (rPⅢ) that displayed T/B combined epitopes. By using either commercial IgG against whole-cell of Helicobacter pylori or rHpaA antiserum as the primary antibody, the T/B combined epitopes displayed in rP Ⅲ s were screened and identified by Western blot and ELISA. MTT was applied to determine the proliferation of rHpaA-immunized mouse splenocytes after stimulation of the different recombinant rPⅢ proteins. Results In the HpaA molecule there were five T/B combined epitopes: HpaA10, HpaA37, HpaA79, HpaA116 and HpaA143. All the T/B combined epitopes were successfully displayed on the surface of PⅢ protein of phage M13. The results of Western blot, ELISA and MTT confirmed that HpaA116 was the predominant antigenic epitope, both HpaA37 and HpaA79 were the efficient antigenic epitopes. However, HpaA10 and HpaA143 were identified as ineffective antigenic epitopes. Conclusion The phage display systems of T/B combined epitope peptides of H. pylori adhesin A have been successfully generated in this study. HpaA37 and HpaA79, especially HpaA116 are the efficient T/B combined antigenic epitopes in HpaA of H. pylori. Key words: Helicobacter pylori; HpaA gene; Phage display; B cell epitope; T cell epitope

Construction of a recombinant attenuatedSalmonella typhimuriumDNA vaccine carryingHelicobacter pylorihpaA

To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then cloned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coli DH5alpha, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.

Prokaryotic expression of fusion gene of vacuolating segment of vacA and hpaA in Helicobacter pylori

AIM: The prokaryotic expression vector of the fusion gene with v segment of the vacuolating cytotoxin and hpaA of Helicobacter pylori (H pylori) was constructed and expressed. It would lay a foundation for prophylaxis and therapy of H pylori infection. METHODS: By using the primer with a fragment encoding 12 amino acids of N-terminal of human interleukin-3 (IL-3), the vacuolating cytotoxin gene of Hp with linker was amplified from pQE30-V plasmid by PCR. The gene was cloned into plasmid pTrc99A-HpaA and fused with the hpaA gene. The fusion gene was cloned into prokaryotic expression vector pQE30. The recombinant plasmid of pQE30-V-HpaA was transformed into E.coli. DH5α and expressed in the presence of IPTG. The expression product was analyzed by SDS-PAGE, its antigenicity of the expression product was identified by Western blotting. RESULTS: Mr of recombinant protein was about 65 000 and represented 35% total protein of E.coli. Western blotting showed the recombinant protein could be recognized by the antiserum against H pylori. CONCLUSION: The fusion gene and its prokaryotic expression vector pQE30-V-HpaA is constructed and expressed in DH5α successfully. It provides the antigen basis for further studying the biological function of fusion protein and obtaining vaccine against the infection of H pylori.

Cloning, expression and identification of hpaA gene from a clinical isolate of Helicobacter pylori

To clone Helicobacter pylori adhesin (hpaA) gene,to construct the expression vector of the gene and to identify immunogenicity of the fusion protein. The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted hpaA gene was constructed. hpaA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein. In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned hpaA gene was from 94.25% approximate, equals 97.32%, while the homology of its putative amino acid sequence was as high as 95.38% approximate, equals 98.46%. The expression output of HpaA fusion protein in pET32a-hpaA-BL21DE3 system was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to preduce high titer antibody after the animal was immunized with the protein. An expression system with high efficiency of H.pylori hpaA gene has been established successfully. The expressed HpaA fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.

Construction of hpaA gene from a clinical isolate of Helicobacter pylori and identification of fusion protein.

To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein. The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E.coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE. Western blot with commercial antibody against whole cell of H.pylori as well as immunodiffusion assay with self-prepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H.pylori and to examine HpaA expression of 109 clinical isolates of H.pylori. In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25-97.32 % and 95.38-98.46 %, respectively. The output of HpaA fusion protein in its expression system of pET32a-hpaA-BL21DE3 was approximately 40 % of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H.pylori and to induce rabbit producing specific antiserum with 1:4 immunodiffusion titer after the animal was immunized with the fusion protein. 81.6 % of the serum samples from 125 patients infected with H.pylori (102/125) were positive for HpaA antibody and all of the tested isolates of H.pylori (109/109) were detectable for HpaA. A prokaryotic expression system with high efficiency of H.pylori hpaA gene was successfully established. The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H.pylori clinical strains and specific antibody production in H.pylori infected patients indicate that HpaA is an excellent and ideal antigen for developing H.pylori vaccine.

Construction of an attenuated Salmonella typhimurium vaccine carrying the Helicobacter pylori hpaA gene

OBJECTIVE: Helicobacter pylori is documented to have infected more than half of the world’s population. It colonizes the human stomach and is associated with the development of chronic active gastritis, peptic ulcer disease, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Immunization against this bacterium represents a cost-effective strategy to reduce global gastric cancer rates and would have a major impact on H. pylori-related peptic ulcer disease. HpaA, a subunit protein of H. pylori adhesin, is known to be the pathogenic factor and attenuated Salmonella typhimurium is a live vaccine vector. The present study aimed at constructing and identifying a live attenuated S. typhimurium vaccine carrying the hpaA gene of H. pylori. METHODS: The hpaA gene was amplified from H. pylori genomic DNA by polymerase chain reaction (PCR) and cloned into the NcoI-SalI site of the prokaryotic expression plasmid pTrc99A. After sequence analysis of the hpaA gene, the identified recombinant plasmid was then used to transform a live attenuated S. typhimurium, namely SL3261, and positive clones were screened by PCR and restriction enzyme digestion. RESULTS: Confirmed by PCR, restriction enzyme digestion and sequence analysis, a recombinant prokaryotic expression plasmid, namely pTrc99A-hpaA, harboring approximately 560 bp hpaA was constructed and the recombinant plasmid was then successfully introduced into a live attenuated S. typhimurium SL3261. CONCLUSION: A recombinant live attenuated S. typhimurium vaccine harboring the H. pylori hpaA gene was constructed and identified. This work will help develop an oral recombinant live vaccine against H. pylori infection.

HpaA shows variable surface localization but the gene expression is similar in different Helicobacter pylori strains

Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in differentH. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno–electron microscopy (IEM), and phase–contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3–4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription–polymerase chain reaction (RT–PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath.

“Cag PAI HAVE A FUNCTION RELATED WITH ADHESION TO GASTRIC EPITHELIAL CELLS”

ObjectiveThe cag pathogenecity island (PAI) is a major virulence factor of Helicobacter pylori (H.p). Andherent ability of H. pylori to the gastric epithelial cells is an important initial step for bacterial virulance. The hpaA and bab A2 genes are thought to be major adherent‐associated genes. Some of cag genes have partial homology to the flagellar aparatus or other adhesion related gene. It is not fully investigated whether cag PAI is related with the adherent ability to the gastric epithelial cells. To evaluate the interaction between the structure of Cag PAI and adherent ability to the gastric epithelial cells.MethodsThe polymerase chain reaction (PCR) and Southern blot analysis were used to determine the structure of cag PAI in Japanese 236 clinical isolates. The PCR and Southern blot analysis were used to verity the presence or absence of hpaA and bab A2. Adherent ability H. pylori to the gastric epithelial cells (KATO cells) was examined in the tested strains by flow‐cytometric analysis (FACS) using anti‐H. pylori antibody.ResultsSeven cag PAI partial deleted strains of tested 236 strains (3.0%) were found by PCR and southern blot analysis, and which showed 3 pattern in deleted region within cag PAI. All cagG‐deleted strains showed less ability of adhesion than CagG positive strains. The peak counts of the cells adhered with cagG deleted strains ( n = 4 ) were significantly lower than that of cagG positive strains ( n = 8 ) ( mean peak count ± SE ; 27.3 ± 2.9 vs 54.8 ± 4.9 ) by FACS analysis. All of cagG‐deleted strains posessed hpaA gene. Six of them possesed babA2 gene.ConclusionsOur results demonstrated that cagG‐deleted clinical isolates had less ability of adhesion to the gastric epithelial cells, even if hpaA and babA2 gene are present.