[show abstract][hide abstract] ABSTRACT: Arginase is an enzyme involved in the last step of the urea cycle, where it catalyses the hydrolysis of L-arginine to generate L-ornithine and urea. Compared to the well-characterised arginases from animals, yeast and other bacteria, Helicobacter pylori arginase, or RocF, is unique in at least three aspects. Firstly, it has been identified as an important factor in evasion of the host's immune system and thus contributes to persistent infection by the bacterium. Secondly, the optimal catalytic conditions of RocF are different from those of other arginases. Finally, sequence alignment indicates that RocF possesses considerable differences at its N- and C-terminal from other arginases and harbours an insertion of 13 residues in the middle of the sequence. To better understand these unique biochemical and enzymatic properties, we therefore have embarked on determining the structure of RocF. In this study, the crystal structure of RocF was solved with the molecular replacement method. Based on the structure and systematic mutagenesis studies, we confirmed that the inserted residues form a helix that was not observed in other arginases and was able to raise the arginase activity by 30% probably by change the conformation of the substrate binding pocket. Six residues were involved in Mn2+ binding, all of which were essential for arginase activity. The C-terminal motif is not sufficient in establishing the oligomeric state of RocF, and no disulphide bonds were observed in RocF.
The international journal of biochemistry & cell biology 02/2013; · 4.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK-motif from M-SAA3 is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process. We used standard solid-phase methods to synthesize a tetrameric branched peptide (sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing 10-mer BP. The aberrant expression of transcription factors (TF) was analyzed using a transcription factor protein/DNA array. MUC3 and relevant TFs were detected using real-time PCR and (or) Western blots. The luciferase assay, EMSA and ChIP assays were used to analyze the activity of the human MUC3 promoter. The bacterial adherence assay was used to evaluate the in vitro inhibition of enteropathogenic Escherichia coli (EPEC) or enterohemorrhagic Escherichia coli serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLK-containing 10-mer BP induced higher levels of MUC3 expression than the M-SAA3 derived N-terminal 10-mer monomeric peptide, and MUC3 expression was activated through transcriptional mechanisms, including the induction of multiple transcription factors and further binding with their cis-elements between nucleotides -242 and -62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP dramatically inhibited EPEC and EHEC O157:H7 adherence to the HT-29-Gal cells compared with the controls. This finding suggests a potential therapeutic use for this peptide to prevent gastrointestinal infection.
Journal of Biological Chemistry 01/2013; · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND & AIMS:: Immunodominance is an important feature of anti-viral, anti-tumor, and anti-bacterial cellular immune responses, but it is not well-demonstrated in the immune responses against Helicobacter pylori (H. pylori). Antigen-specific CD4(+) T cells protect mice against infection with H. pylori. We investigated the immunodominant CD4(+) T-cell response to neuraminyllactose-binding hemagglutinin (HpaA)- a conserved, H. pylori -specific colonization factor that is being investigated as an antigen for vaccination strategies. METHODS:: HpaA-specific CD4(+) T cells were expanded with autologous peripheral blood mononuclear cells (PBMCs) that had been incubated with recombinant HpaA, and characterized using overlapping synthetic peptides. We compared the percentage of CD4(+) T cells with specificity for HpaA(88-100), restricted to HLA-DRB1*1501, among 59 H. pylori -infected subjects with different gastric diseases. RESULTS:: We identified and characterized several immunodominant CD4(+) T cell epitopes derived from HpaA. The immunodominant CD4(+) T-cell responses specific to HpaA(88-100) were observed in most H. pylori -infected individuals who expressed HLA-DRB1*1501, and were significantly more abundant in patients with less severe diseases ( P <0.05). CONCLUSIONS:: The HLA-DRB1*1501-restricted immunodominant CD4(+) T-cell response to HpaA(88-100) is associated with reduced risk of severe gastric diseases. Further study of these and other immunodominant CD4(+) T-cell responses to H. pylori will provide insight into mechanisms of protective immunity and aide in vaccine design.
[show abstract][hide abstract] ABSTRACT: Most of the Helicobacter pylori strains containing the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI is composed of 27 proteins, and some of the components are required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8); however, the exact function of most of the components remains unknown or poorly characterized. In this study, we demonstrated that CagT (HP0532), which is an essential structural component of the cag PAI apparatus, plays an important role in the translocation of CagA into host epithelial cells. In addition to being located on the bacterial surface, CagT is also partially localized in the inner membrane, where it acts as a chaperone-like protein and promotes CagA translocation. However, CagT secretion was not detected by immunoprecipitation analysis of cell culture supernatants. Meanwhile, CagT was related to the introduction of IL-8 of the host cell. These results suggest that CagT is expressed on both the inner and outer bacterial membranes, where it serves as a unique type IV secretion system component that is involved in CagA secretion and cag PAI apparatus assembly.
Journal of Microbiology and Biotechnology 10/2012; 22(10):1343-9. · 1.40 Impact Factor
[show abstract][hide abstract] ABSTRACT: Octaprenyl pyrophosphate synthase (OPPs), an enzyme belonging to the trans-prenyltransferases family, is involved in the synthesis of C40 octaprenyl pyrophosphate (OPP) by reacting farnesyl pyrophosphate (FPP) with five isopentenyl pyrophosphates (IPP). It has been reported that OPPs is essential for bacteria's normal growth and is a potential target for novel antibacterial drug design. Here we report the crystal structure of OPPs from Helicobacter pylori, determined by MAD method at 2.8Å resolution and refined to 2.0Å resolution. The substrate IPP was docked into HpOPPs structure and residues involved in IPP recognition were identified. The other substrate FPP, the intermediate GGPP and a nitrogen-containing bisphosphonate drug were also modeled into the structure. The resulting model shed some lights on the enzymatic mechanism, including (1) residues Arg87, Lys36 and Arg39 are essential for IPP binding; (2) residues Lys162, Lys224 and Gln197 are involved in FPP binding; (3) the second DDXXD motif may involve in FPP binding by Mg(2+) mediated interactions; (4) Leu127 is probably involved in product chain length determination in HpOPPs and (5) the intermediate products such as GGPP need a rearrange to occupy the binding site of FPP and then IPP is reloaded. Our results also indicate that the nitrogen-containing bisphosphonate drugs are potential inhibitors of FPPs and other trans-prenyltransferases aiming at blocking the binding of FPP.
The international journal of biochemistry & cell biology 09/2012; 44(12):2116-2123. · 4.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tetanus, a severe infectious disease, is caused by tetanus toxin (TT) from Clostridium tetani, which remains one of the most critical unsolved health problems despite preventive strategies. The carboxyl terminal of TT (TTC) is responsible for the binding of TT to neurons and for its toxicity and has been proven to be immunogenic and protective in various forms. It would therefore be extremely interesting to identify the epitope on TTC at a molecular level. In this study, we generated a neutralizing monoclonal antibody, 5C4, which inhibited TT binding to its receptor and was efficiently protective at 73.7IU/mg. Moreover, 5C4 recognized a novel linear epitope on TT, namely TC((1155-1171)), which spans from Lys1155 to Val1171. In addition, TC((1155-1171)) was shown to elicit the production of a serum IgG that protected mice against a challenge with TT. These results suggested that TC((1155-1171)) and the monoclonal antibody 5C4 are good candidates for the development of epitope-based vaccines and therapeutic antibodies against tetanus.
[show abstract][hide abstract] ABSTRACT: The infection rate of Helicobacter pylori is high all over the world, especially in the Chinese Tibetan Plateau. Here, we report the genome sequence of Helicobacter pylori strain XZ274 isolated from a Tibetan patient with gastric cancer. The strain contains 1,634,138 bp with 1,654 coding sequences and a pXZ274 plasmid of 22,406 bp with 26 coding sequences. This is the first complete genome sequence of Helicobacter pylori from the Tibetan Plateau in China.
Journal of bacteriology 08/2012; 194(15):4146-7. · 3.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although Th22 and Th17 cells have been reported to play critical roles during autoimmunity and inflammation, information on their role in cancer-immunity is limited. In this study, we investigated clinical relevance of circulating Th22 and Th17 cells in patients with gastric cancer (GC). Using multi-color flow cytometry and PMA stimulation, we determined the levels of Th22, Th17 and Th1 cells in the peripheral blood of 32 GC patients and 19 healthy donors, and evaluated their correlations with tumor stage and overall survival. Compared with healthy donors, the frequencies of circulating CD4(+)IL-22(+) T cells, CD4(+)IL-17(+) T cells, Th22 (CD4(+)IL-22(+)IL-17(-)INF-γ(-)) cells, Th17 (CD4(+)IL-17(+)INF-γ(-)) cells were increased in patients with GC, but there was no significant differences in the frequencies of CD4(+)IFN-γ(+) T cells and Th1 (CD4(+)IL-17(-)INF-γ(+)) cells. Th22 cells showed positive correlation with Th17 cells and CD4(+)IL-17(+) T cells in patients with GC. Furthermore, the frequencies of Th22 and Th17 cells were significantly higher in stage III-IV GC patients versus stage I-II and correlated with patients' overall survival. These data suggest that circulating Th22 cells as well as Th17 cells are increased in the peripheral blood of GC patients with tumor progression, and that these cells may be promising novel clinical markers for GC.
Journal of Clinical Immunology 07/2012; · 3.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: ThyX, a flavin-dependent thymidylate synthase that is involved in the synthesis of dTMP from dUMP, is a promising target for the development of novel antibacterial drugs that aimed at blocking the biosynthesis of dTMP, one of the building blocks of DNA. This enzyme has been recently identified in some dsDNA viruses and pathogenic bacteria, including the gastric pathogen Helicobacter pylori. It shares neither sequence nor structural homology with the classical ThyA in humans and other organisms. Further more, ThyX and ThyA are the only source of dTMP in these organisms and other pathways cannot substitute for their function. Thus, ThyX-specific inhibitors could be effective antibacterial reagents while having no impact on human cells. Here we report the crystal structure of ThyX from Helicobacter pylori strain 26695 in complex with co-factor FAD and substrate dUMP at 2.5 A resolution, which consists of a 1.5 tetramer of ThyX with a total of 1248 residues, six FAD and six dUMP molecules in an asymmetric unit. The structure revealed the key residues that are involved in co-factor FAD and substrate dUMP binding, site-directed mutagenesis were performed to analysis the importance of these residues on ThyX activity by genetic complementation and FAD binding assay.
Protein and Peptide Letters 04/2012; 19(11):1225-30. · 1.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: The stability of protein drugs remains one of the key hurdles to their success in the market. The aim of the present study was to design a novel nanoemulsion drug-delivery system (NEDDS) that would encapsulate a standard-model protein drug - bovine serum albumin (BSA) - to improve drug stability.
The BSA NEDDS was prepared using a phase-inversion method and pseudoternary phase diagrams. The following characteristics were studied: morphology, size, zeta potential, drug loading, and encapsulation efficiency. We also investigated the stability of the BSA NEDDS, bioactivity of BSA encapsulated within the NEDDS, the integrity of the primary, secondary, and tertiary structures, and specificity.
The BSA NEDDS consisted of Cremophor EL-35, propylene glycol, isopropyl myristate, and normal saline. The average particle diameter of the BSA NEDDS was about 21.8 nm, and the system showed a high encapsulation efficiency (>90%) and an adequate drug-loading capacity (45 mg/mL). The thermodynamic stability of the system was investigated at different temperatures and pH levels and in room-temperature conditions for 180 days. BSA NEDDS showed good structural integrity and specificity for the primary, secondary, and tertiary structures, and good bioactivity of the loaded BSA.
BSA NEDDS showed the properties of a good nanoemulsion-delivery system. NEDDS can greatly enhance the stability of the protein drug BSA while maintaining high levels of drug bioactivity, good specificity, and integrity of the primary, secondary, and tertiary protein structures. These findings indicate that the nanoemulsion is a potential formulation for oral administration of protein drugs.
International Journal of Nanomedicine 01/2012; 7:5529-43. · 3.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: The heme oxygenase ChuZ is part of the iron acquisition mechanism of Campylobacter jejuni, a major pathogen causing enteritis in humans. ChuZ is required for C. jejuni to use heme as the sole iron source. The crystal structure of ChuZ was resolved at 2.5Å, and it was revealed to be a homodimer with a split-barrel fold. One heme-binding site was at the dimer interface and another novel heme-binding site was found on the protein surface. Heme was bound in this site by four histidine side-chains through hydrophobic interactions. Based on stoichiometry studies and comparisons with other proteins, the possibility that similar heme-binding site exists in homologous proteins and its possible functions are discussed. The structural and mutagenesis analyses reported here establish ChuZ and ChuZ homologs as a new bacterial heme oxygenase family apart from the canonical and IsdG/I families. Our studies provide insight into the enzymatic mechanisms and structure-function relationship of ChuZ.
Biochemical and Biophysical Research Communications 11/2011; 415(1):82-7. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: There is an urgent need for vaccine against enterohemorrhagic Escherichia coli (EHEC), which causes a wide range of life-threatening diseases in human and animals. E. coli secreted protein A (EspA), intimin and shiga toxin (Stx) are important pathogenic factors and protective antigens of EHEC. In our previous study, we found that recombinant trivalent protein EIS, which is composed of EspA (E), the 300 amino acids of the carboxyl terminus of intimin (I) and the B subunit of Stx2 (S), was able to efficiently elicit protective immunity against EHEC. The application of live attenuated Salmonella as a carrier for vaccine against mucosal pathogens provided unparalleled merits. Therefore, in this study we constructed live attenuated EIS-producing Salmonella vaccine and tested it as vaccine in mice model. We found that the vaccination of EIS-producing recombinant Salmonella was able to induce significant increases of EspA, intimin and Stx2 specific IgG in serum and secretory IgA in feces. Antigen specific T cell proliferation was also observed in the mice immunized with recombinant EIS-producing Salmonella. In addition, this immunity was able to protect mice from a challenge of a lethal dose of EHEC, even after a period of 70 days. Moreover, the EIS-producing Salmonella induced immunity can be boosted by a single subcutaneous injection of purified EIS protein, even after an interval of 70 days. This EIS-producing Salmonella vaccine provides an alternative approach for the prevention of EHEC infection.
[show abstract][hide abstract] ABSTRACT: Helicobacter pylori arginase is an important factor in evasion of the host's immune system and contributes to persistent infection by this bacterium. It is unique in many aspects compared with other arginases: for example, it has optimal activity with Co(2+) as a cofactor rather than Mn(2+) and has strongest activity at acidic pH instead of alkaline pH. In this study, H. pylori arginase was purified and crystallized in complex with Mn(2+) and a diffraction data set was collected to 2.2 Å resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 94.69, b = 102.24, c = 148.61 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 06/2011; 67(Pt 6):707-9. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Octaprenyl pyrophosphate synthase (OPPs) is involved in the synthesis of the side chains of ubiquinone and menaquinone and catalyzes consecutive condensation reactions of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate polyprenyl pyrophosphate and pyrophosphate. In order to investigate the roles played by OPPs in the metabolism of ubiquinone and menaquinone and the enzymatic mechanisms of these enzymes, analysis of the structure-function relationship of OPPs from Helicobacter pylori was initiated. The gene for OPPs was cloned, the protein was expressed, purified and crystallized and a diffraction data set was collected to 2.00 Å resolution. The crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 109.33, c = 103.41 Å.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 02/2011; 67(Pt 2):263-5. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Detection of Shiga toxin-producing Escherichia coli O157:H7 from commercially grown pigs has been reported. Furthermore, the E. coli O157:H7 colonized model of pig has been established and E. coli O157:H7 could be transmitted from infected donor pigs to naïve pigs directly and indirectly. In the present study, we want to know whether any E. coli O157:H7 strain can colonize to the alimentary tract of pig and the virulence of E. coli O157:H7 to pig by injection. Bama miniature pig was infected with E. coli O157:H7 EDL933 strain orally, but the organism could not be recovered from the feces and did not cause any tissue damage. Nevertheless, this pathogen introduced serious clinical symptoms and pathological injuries by injection, especially the nervous system and the injected pig exhibited severe neurological symptoms, including synclonus tremens, ataxia, head-pressing and recumbency, etc. The pig did not excrete urine and feces and the abdomen became tympanous. These data suggested that only certain E. coli O157:H7 strains could colonize to the GIs of pigs involved mechanisms that related to various factors. However, the organism has strong virulence to pig by injection mode and it is a risky pathogen to human health.
African journal of microbiology research 01/2011; 4:2461-2467. · 0.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Arginase, a manganese-dependent enzyme that widely distributed in almost all creatures, is a urea cycle enzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. Compared with the well-studied arginases from animals and yeast, only a few eubacterial arginases have been characterized, such as those from H. pylori and B. anthracis. However, these enzymes used for arginase activity assay were all expressed with LB medium, as low concentration of Mn(2+) was detectable in the medium, protein obtained were partially Mn(2+) bonded, which may affect the results of arginase activity assay. In the present study, H. pylori arginase (RocF) was expressed in a Mn(2+) and Co(2+) free minimal medium, the resulting protein was purified through affinity and gel filtration chromatography and the apo-form of RocF was confirmed by flame photometry analysis. Gel filtration indicates that the enzyme exists as monomer in solution, which was unique as compared with homologous enzymes. Arginase activity assay revealed that apo-RocF had an acidic pH optimum of 6.4 and exhibited metal preference of Co(2+)>Ni(2+)>Mn(2+). We also confirmed that heat-activation and reducing regents have significant impact on arginase activity of RocF, and inhibits S-(2-boronoethyl)-L-Cysteine (BEC) and Nω-hydroxy-nor-Arginine (nor-NOHA) inhibit the activity of RocF in a dose-dependent manner.
PLoS ONE 01/2011; 6(10):e26205. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a primarily food-borne bacterial pathogen that is capable of causing life-threatening human infections and poses a serious challenge to public health worldwide. The bacterial outer-membrane protein intimin plays a key role in the initiation process of EHEC infection. In this study, intimin from EHEC O157:H7 (Int188) and its N916Y mutant (IntN916Y) were purified and crystals of both were obtained using the hanging-drop vapour-diffusion method at 291 K. Data were collected from Int188 and IntN916Y crystals to 2.8 and 2.6 Å resolution, respectively. The crystal of Int188 belonged to the orthorhombic space group C2, with unit-cell parameters a=235.16, b=44.81, c=129.12 Å, α=γ=90, β=97.53°. The crystal of IntN916Y belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=43.78, b=92.49, c=100.05 Å, α=β=γ=90°.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2011; 67(Pt 1):41-3. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His(245) and an azide molecule when it is present in crystallization conditions. Experiments show that Arg(166), which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His(245), surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.
Journal of Biological Chemistry 10/2010; 286(2):1537-44. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Helicobacter pylori (H. pylori) is a major human pathogenic bacterium in gastric mucosa. However, the regulatory mechanism of H. pylori-induced immune response is not clear. MicroRNAs (miRNAs) have recently emerged as key post-transcriptional regulators of gene expression, and their role in H. pylori infection is just beginning to be explored. Here, we first reported that H. pylori infection up-regulated the expression of miR-146a in gastric epithelial cells as well as in gastric mucosal tissues in NF-κB-dependent manner. In turn, miR-146a may downregulate the expression of target genes, interleukin-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6). Furthermore, miR-146a negatively regulated H. pylori-triggered interleukin (IL)-8, growth-related oncogene (GRO)-α, and macrophage inflammatory protein (MIP) -3α through diminishing NF-κB activity. In conclusion, H. pylori-induced miR-146a plays a potential role in a negative feedback loop to modulate the inflammation by targeting IRAK1 and TRAF6.
Microbes and Infection 10/2010; 12(11):854-63. · 2.92 Impact Factor