Phenylphosphonate transport by Helicobacter pylori

BACKGROUND: Helicobacter pylori can utilize phenylphosphonate as a sole source of phosphorus, and it is able to transport the phosphonate N-phosphonoacetyl-L-aspartate. However, H. pylori does not have any genes homologous to those of the known pathways for phosphonate degradation in bacteria, indicating that it must have novel pathways for the transport and metabolism of phosphonates. METHODS: Phenylphosphonate transport by H. pylori was studied in strains LC20, J99 and N6 by the centrifugation through oil method using [(14)C]-labeled phenylphosphonate. RESULTS: The Michaelis constants of transport K(t) and V(max) for phenylphosphonate showed similar kinetics in the three strains. The Arrhenius plot for phenylphosphonate transport rates at permeant concentrations of 50 micromol/L was linear over the temperature range 10-40 degrees C with an activation energy of 3.5 kJ/mol, and a breakpoint between 5 and 10 degrees C. Transport rates increased with monovalent cation size. The effects of various inhibitors were investigated: iodoacetamide, amiloride, valinomycin, and nigericin reduced the rate of phenylphosphonate transport; sodium azide and sodium cyanide increased the transport rate; and monensin had no effect. CONCLUSIONS: The kinetics and properties of H. pylori phenylphosphonate transport were characterized, and the data suggested a carrier-mediated transport mechanism.     

Characterization of arginine transport in Helicobacter pylori

Background. The amino acid L‐arginine is an essential requirement for growth of Helicobacter pylori. Several physiological roles of this amino acid have been identified in the bacterium, but very little is known about the transport of L‐arginine and of other amino acids into H. pylori. Methods. Radioactive tracer techniques using L‐(U‐¹⁴C) arginine and the centrifugation through oil method were employed to measure the kinetic parameters, temperature dependence, substrate specificity, and effects of analogues and inhibitors on L‐arginine transport. Results. The transport of arginine at millimolar concentrations was saturable with a K_m of 2.4 ± 0.3 mM and V_max of 1.3 ± 0.2 pmole min^?1 (µl cell water)^?1 or 31 ± 3 nmole per minute (mg protein)^?1 at 20°C, depended on temperature between 4 and 40°C, and was susceptible to inhibitors. These characteristics suggested the presence of one or more arginine carriers. The substrate specificity of the transport system was studied by measuring the effects of L‐arginine analogues and amino acids on the rates of transport of L‐arginine. The absence of inhibition in competition experiments with L‐lysine and L‐ornithine indicated that the transport system was not of the Lysine‐Arginine‐Ornithine or Arginine‐Ornithine types. The presence of different monovalent cations did not affect the transport rates. Several properties of L‐arginine transport were elucidated by investigating the effects of potential inhibitors. Conclusions. The results provided evidence that the transport of L‐arginine into H. pylori cells was carrier‐mediated transport with the driving force supplied by the chemical gradient of the amino acid.     

The Helicobacter pylori CagA protein induces tyrosine dephosphorylation of ezrin

Helicobacter pylori is one of the most wide-spread bacterial pathogens and infects the human stomach to cause diseases, such as gastritis, gastric ulceration, and gastric cancer. A major virulence determinant is the H. pylori CagA protein (encoded by the cytotoxin-associated gene A) which is translocated from the bacteria into the cytoplasm of host cells by a type IV secretion system. In the host cell, CagA is phosphorylated on tyrosine residues and induces rearrangements of the actin cytoskeleton. We have previously shown that tyrosine-phosphorylated CagA inhibits the catalytic activity of Src family kinases and induces tyrosine dephosphorylation of several host cell proteins. Here, we identified one of these proteins as ezrin by a combination of preparative gel electrophoresis, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Specific pharmacological inhibition of Src family kinases also induces ezrin dephosphorylation. Therefore, ezrin dephosphorylation appears to be induced by CagA-mediated Src inactivation. Ezrin is the founding member of the ezrin-radixin-moesin (ERM) family of proteins which are signalling integrators at the cell cortex. Since ezrin is a component of microvilli and a linker protein between actin filaments and membrane proteins, this observation has important implications for H. pylori pathogenesis and might also help to explain the development of gastric cancer.     

Structural insight into the distinct properties of copper transport by the Helicobacter pylori CopP protein

Helicobacter pylori CopP (HpCopP) is a putative copper binding regulatory protein composed of 66 amino acid residues. The small HpCopP protein is homologous to CopZ, encoded by the E. hirae and B. subtilis cop operons. To clarify the role of HpCopP in copper metabolism in H. pylori, we studied the structural and copper binding characteristics by NMR spectroscopy. Based on the resonance assignments, the tertiary structure of HpCopP was determined. Unlike the betaalphabetabetaalphabeta fold of the homologous CopZ, HpCopP adopts the betaalphabetabetaalpha fold. The superposition with structures of other bacterial copper binding proteins showed that the global structure of HpCopP follows the general topology of the family, regardless of absence of the C-terminal beta-strand. The Cu(I) binding property of HpCopP was well conserved like CopZs: the structural changes due to Cu(I) and Ag(I) bindings were primarily restricted to the metal binding motif (CXXC motif). On the other hand, the Cu(II) binding property of CopP was different with that of CopZ: in the absence of reducing agent, Cu(II) ion oxidized a mutant HpCopP, resulting in disulfide bond formation in the CXXC motif. The Cu(II) ion binding property was evaluated using the mutant HpCopP, in which two amino acids were artificially introduced at the C-terminus, since the reduced state of the CXXC motif was more stabile in the mutant HpCopP without a reducing agent. Here, the structure and copper binding property of HpCopP are discussed in detail.     

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.     

A novel apoptosis-inducing protein from Helicobacter pylori

Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori-mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited gamma-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-l-norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial gamma-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant gamma-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that gamma-glutamyl transpeptidase plays a significant role in H. pylori-mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial gamma-glutamyl transpeptidase function.     

The cysteine-rich protein A from Helicobacter pylori is a beta-lactamase

Among the large number of hypothetical proteins within the genomes of Helicobacter pylori, there is a family of unique and highly disulfide-bridged proteins, designated family 12, for which no function could originally be assigned. Sequence analysis revealed that members of this family possess a modular architecture of alpha/beta-units and a stringent pattern of cysteine residues. The H. pylori cysteine-rich protein A (HcpA), which is a member of this family, was expressed and refolded from inclusion bodies. Six pairs of cysteine residues, which are separated by exactly seven residues, form disulfide bridges. HcpA is a beta-lactamase. It slowly hydrolyzes 6-aminopenicillinic acid and 7-aminocephalosporanic acid (ACA) derivatives. The turnover for 6-aminopenicillinic acid derivatives is 2-3 times greater than for ACA derivatives. The enzyme is efficiently inhibited by cloxacillin and oxacillin but not by ACA derivatives or metal chelators. We suggest that all family 12 members possess similar activities and might be involved in the synthesis of the cell wall peptidoglycan. They might also be responsible for amoxicillin resistance of certain H. pylori strains.     

Structure of the neutrophil-activating protein from Helicobacter pylori

Helicobacter pylori is a major human pathogen associated with severe gastroduodenal diseases, including ulcers and cancers. An H.pylori protein that is highly immunogenic in humans and mice has been identified recently. This protein has been termed HP-NAP, due to its ability of activating neutrophils. In order to achieve a molecular understanding of its unique immunogenic and pro-inflammatory properties, we have determined its three-dimensional structure. Its quaternary structure is similar to that of the dodecameric bacterial ferritins (Dps-like family), but it has a different surface potential charge distribution. This is due to the presence of a large number of positively charged residues, which could well account for its unique ability in activating human leukocytes.     

The Helicobacter pylori 19.6-kilodalton protein is an iron-containing protein resembling ferritin.

The gastric pathogen Helicobacter pylori has been shown to produce a 19.6-kDa protein with apparent binding activity for erythrocytes, human buccal epithelial cells, and laminin. In this report we demonstrate that it is an iron-binding protein, resembling ferritin both structurally and biochemically. Also, because its binding activity for laminin, erythrocytes, and buccal cells was abolished by low concentrations of Tween 20, binding is likely nonspecific.     

The Helicobacter pylori MutS protein confers protection from oxidative DNA damage

The human gastric pathogenic bacterium Helicobacter pylori lacks a MutSLH-like DNA mismatch repair system. Here, we have investigated the functional roles of a mutS homologue found in H. pylori, and show that it plays an important physiological role in repairing oxidative DNA damage. H. pylori mutS mutants are more sensitive than wild-type cells to oxidative stress induced by agents such as H2O2, paraquat or oxygen. Exposure of mutS cells to oxidative stress results in a significant ( approximately 10-fold) elevation of mutagenesis. Strikingly, most mutations in mutS cells under oxidative stress condition are G:C to T:A transversions, a signature of 8-oxoguanine (8-oxoG). Purified H. pylori MutS protein binds with a high specific affinity to double-stranded DNA (dsDNA) containing 8-oxoG as well as to DNA Holliday junction structures, but only weakly to dsDNA containing a G:A mismatch. Under oxidative stress conditions, mutS cells accumulate higher levels (approximately threefold) of 8-oxoG DNA lesions than wild-type cells. Finally, we observe that mutS mutant cells have reduced colonization capacity in comparison to wild-type cells in a mouse infection model.     

The VacA toxin of Helicobacter pylori identifies a new intermediate filament-interacting protein

The VacA toxin produced by Helicobacter pylori acts inside cells and induces the formation of vacuoles arising from late endosomal/lysosomal compartments. Using VacA as bait in a yeast two-hybrid screening of a HeLa cell library, we have identified a novel protein of 54 kDa (VIP54), which interacts specifically with VacA, as indicated by co-immunoprecipitation and binding experiments. VIP54 is expressed in cultured cells and many tissues, with higher expression in the brain, muscle, kidney and liver. Confocal immunofluorescence microscopy with anti-VIP54 affinity- purified antibodies shows a fibrous pattern typical of intermediate filaments. Double label immunofluorescence performed on various cell lines with antibodies specific to different intermediate filament proteins revealed that VIP54 largely co-distributes with vimentin. In contrast to known intermediate filament proteins, VIP54 is predicted to contain approximately 50% of helical segments, but no extended coiled-coil regions. The possible involvement of this novel protein in interactions between intermediate filaments and late endosomal compartments is discussed.     

DNA-binding activity of TNF-alpha inducing protein from Helicobacter pylori

Tumor necrosis factor-alpha (TNF-alpha) inducing protein (Tipalpha) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-alpha and chemokine genes and activation of nuclear factor-kappaB. Since Tipalpha enters gastric cancer cells, the Tipalpha binding molecules in the cells should be investigated. The direct DNA-binding activity of Tipalpha was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tipalpha and DNA, revealed that the affinity of Tipalpha for (dGdC)10 is 2400 times stronger than that of del-Tipalpha, an inactive Tipalpha. This suggests a strong correlation between DNA-binding activity and carcinogenic activity of Tipalpha. And the DNA-binding activity of Tipalpha was first demonstrated with a molecule secreted from H. pylori.     

Potentiation of Helicobacter pylori CagA protein virulence through homodimerization

Chronic infection with Helicobacter pylori cagA-positive strains is associated with atrophic gastritis, peptic ulceration, and gastric carcinoma. The cagA gene product, CagA, is delivered into gastric epithelial cells via type IV secretion, where it undergoes tyrosine phosphorylation at the EPIYA motifs. Tyrosine-phosphorylated CagA binds and aberrantly activates the oncogenic tyrosine phosphatase SHP2, which mediates induction of elongated cell morphology (hummingbird phenotype) that reflects CagA virulence. CagA also binds and inhibits the polarity-regulating kinase partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) via the CagA multimerization (CM) sequence independently of tyrosine phosphorylation. Because PAR1 exists as a homodimer, two CagA proteins appear to be passively dimerized through complex formation with a PAR1 dimer in cells. Interestingly, a CagA mutant that lacks the CM sequence displays a reduced SHP2 binding activity and exhibits an attenuated ability to induce the hummingbird phenotype, indicating that the CagA-PAR1 interaction also influences the morphological transformation. Here we investigated the role of CagA dimerization in induction of the hummingbird phenotype with the use of a chemical dimerizer, coumermycin. We found that CagA dimerization markedly stabilizes the CagA-SHP2 complex and thereby potentiates SHP2 deregulation, causing an increase in the number of hummingbird cells. Protrusions of hummingbird cells induced by chemical dimerization of CagA are further elongated by simultaneous inhibition of PAR1. This study revealed a role of the CM sequence in amplifying the magnitude of SHP2 deregulation by CagA, which, in conjunction with the CM sequence-mediated inhibition of PAR1, evokes morphological transformation that reflects in vivo CagA virulence.     

幽门螺杆菌空泡毒素研究进展

幽门螺杆菌空泡毒素是该菌产生的已知其它细菌毒素无明显源性的唯一蛋白毒素。该毒素是幽门螺杆菌重要的毒力致病因子,它的产生与感染胃肠上皮损伤和溃疡形成密切相关。本就幽门螺杆菌空泡毒素的结构与功能研究进展以及在未来免疫预防与免疫治疗中的作用进行了阐述。     

The secE gene of Helicobacter pylori

Despite extensive annotation by two independent teams, the Helicobacter pylori genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing secE component of the major secretion machinery of Helicobacter pylori.     

The thioredoxin system of Helicobacter pylori

This paper describes the purification of thioredoxin reductase (TR) and the characterization, purification, and cloning of thioredoxin (Trx) from Helicobacter pylori. Purification, amino acid sequence analysis, and molecular cloning of the gene encoding thioredoxin revealed that it is a 12-kDa protein which possesses the conserved redox active motif CGPC. The gene encoding Trx was amplified by polymerase chain reaction and inserted into a pET expression vector and used to transform Escherichia coli. Trx was overexpressed by induction with isopropyl-1-thio-beta-D-galactopyranoside as a decahistidine fusion protein and was recovered from the cytoplasm as a soluble and active protein. The redox activity of this protein was characterized using several mammalian proteins of different architecture but all containing disulfide bonds. H. pylori thioredoxin efficiently reduced insulin, human immunoglobulins (IgG/IgA/sIgA), and soluble mucin. Subcellular fractionation analysis of H. pylori revealed that thioredoxin was associated largely with the cytoplasm and inner membrane fractions of the cell in addition to being recovered in the phosphate-buffered saline-soluble fraction of freshly harvested cells. H. pylori TR was purified to homogeneity by chromatography on DEAE-52, Cibacron blue 3GA, and 2',5'-ADP-agarose. Gel filtration revealed that the native TR had a molecular mass of 70 kDa which represented a homodimer composed of two 35-kDa subunits, as determined by SDS-polyacrylamide gel electrophoresis. H. pylori TR (NADPH-dependent) efficiently catalyzed the reduction of 5,5'-dithiobis(nitrobenzoic acid) in the presence of either native or recombinant H. pylori Trx. H. pylori Trx behaved also as a stress response element as broth grown bacteria secreted Trx in response to chemical, biological, and environmental stresses. These observations suggest that Trx may conceivably assist H. pylori in the process of colonization by inducing focal disruption of the oligomeric structure of mucin while rendering host antibody inactive through catalytic reduction.     

The vacuolating cytotoxin of Helicobacter pylori

Helicobacter pylori, the causative agent of chronic superficial gastritis and duodenal ulcer disease in humans, produces a unique cytotoxin (VacA) that induces cytoplasmic vacuolation in eukaryotic cells. The structural organization and processing of the vacuolating cytotoxin are characteristic of a family of proteins exemplified by Neisseria gonorrhoeae IgA protease. Although only 50% of H. pylori isolates produce detectable cytotoxin activity in vitro, vacA homologues are present in virtually all isolates. Several families of vacA alleles have been identified, and there is a strong correlation between presence of specific vacA genotypes, cytotoxin activity, and peptic ulceration. Experiments in a mouse model of H. pylori-induced gastric damage indicate that the cytotoxin plays an important role in inducing gastric epithelial necrosis.     

Electrogenic nature of rat sodium-dependent multivitamin transport

We report on the electrogenic nature of the transport process mediated by the rat sodium-dependent multivitamin transporter. In Cos-7 cells, the relationship of Na(+) concentration versus biotin and pantothenate uptake rate was sigmoidal with a Na(+):substrate stoichiometry of 2:1. In Cos-7 cells expressing rat SMVT biotin transport was significantly higher when the membrane was hyperpolarized and considerably reduced when the membrane was depolarized. Similarly, biotin uptake in X. laevis oocytes expressing rat SMVT was inhibited with depolarized oocyte membrane by altering the K(+) permeability across the membrane. It is concluded that the transport of biotin and pantothenate mediated by rat SMVT is electrogenic with a Na(+):substrate coupling ratio of 2:1 and that the transport process is associated with the transfer of one net positive charge across the membrane per transport cycle.