Wen-Ching Wang

Cholesterol Depletion Reduces Helicobacter pylori CagA Translocation and CagA-Induced Responses in AGS Cells

ABSTRACT Infection with Helicobacter pylori cagA-positive strains is associated with gastritis, ulcerations, and gastric cancer. CagA is translocated into infected epithelial cells by a type IV secretion system and can be tyrosine phosphorylated, inducing signal transduction and motogenic responses in epithelial cells. Cellular cholesterol, a vital component of the membrane, contributes to membrane dynamics and functions and is important in VacA intoxication and phagocyte evasion during H. pylori infection. In this investigation, we showed that cholesterol extraction by methyl-β-cyclodextrin reduced the level of CagA translocation and phosphorylation. Confocal microscope visualization revealed that a significant portion of translocated CagA was colocalized with the raft marker GM1 and c-Src during infection. Moreover, GM1 was rapidly recruited into sites of bacterial attachment by live-cell imaging analysis. CagA and VacA were cofractionated with detergent-resistant membranes (DRMs), suggesting that the distribution of CagA and VacA is associated with rafts in infected cells. Upon cholesterol depletion, the distribution shifted to non-DRMs. Accordingly, the CagA-induced hummingbird phenotype and interleukin-8 induction were blocked by cholesterol depletion. Raft-disrupting agents did not influence bacterial adherence but did significantly reduce internalization activity in AGS cells. Together, these results suggest that delivery of CagA into epithelial cells by the bacterial type IV secretion system is mediated in a cholesterol-dependent manner.

Vacuolating Toxin Production in Clinical Isolates of Helicobacter pylori with Different vacA Genotypes

A vacuolating cytotoxin encoded by vacA in Helicobacter pylori is known as a potential virulent determinant. The relationship between different vacA alleles, vacuolating ability, and H. pylori-related diseases was investigated. Genetic analysis of 119 isolates from Taiwanese patients revealed that 104 strains were s1a/m2, 13 strains were characterized as the s1a/m1T type, which was more homologous to the s1a/m1 strains, and 2 were characterized as the s1a/m1Tm2 chimeric type. Production of high-grade cytotoxin among 11 strains with s1a/m1T was higher (72.7%) than among 66 strains with s1a/m2 (21.2%) (P < .01). Peptic ulcer occurred in 76.9% of 13 patients with s1a/m1T strains compared with 46.2% of 104 patients with s1a/m2 strains (P < .05). These results suggest that s1a/m1T strains are associated with increased cytotoxic activity and higher ulcer prevalence than are s1a/m2 strains.

Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.

Primary resistance to antibiotics and its clinical impact on the efficacy of Helicobacter pylori lansoprazole-based triple therapies

To evaluate Helicobacter pylori primary resistance and its clinical impact on the efficacy of two lansoprazole‐based eradication triple therapies.

High Prevalence of cagA- and babA2-Positive Helicobacter pylori Clinical Isolates in Taiwan

ABSTRACT Two virulence markers, cagA and babA2, were characterized by PCR in 101 Helicobacter pylori isolates from a population in Taiwan. cagA was detected in 99% of the isolates, while babA2 was present in all of the isolates. Base deletions and substitutions at the forward babA2 primer annealing sites were found. Given their high prevalence, cagA and babA2 cannot be useful markers for predicting the high-risk patients of H. pylori infection in Taiwan.

Binding and internalization of Helicobacter pylori VacA via cellular lipid rafts in epithelial cells.

In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells. Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity. Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it. Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes. Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line. Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules. These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.

Structural Basis for Shikimate-Binding Specificity of Helicobacter pylori Shikimate Kinase

Shikimate kinase (EC 2.7.1.71) catalyzes the specific phosphorylation of the 3-hydroxyl group of shikimic acid in the presence of ATP. As the fifth key step in the shikimate pathway for aromatic amino acid biosynthesis in bacteria, fungi, and plants, but not mammals, shikimate kinase represents an attractive target for the development of new antimicrobial agents, herbicides, and antiparasitic agents. Here, we report the 1.8-Angstroms crystal structure of Helicobacter pylori shikimate kinase (HpSK). The crystal structure shows a three-layer alpha/beta fold consisting of a central sheet of five parallel beta-strands flanked by seven alpha-helices. An HpSK-shikimate-PO(4) complex was also determined and refined to 2.3 Angstroms, revealing induced-fit movement from an open to a closed form on substrate binding. Shikimate is located above a short 3(10) helix formed by a strictly conserved motif (GGGXV) after beta(3). Moreover, several highly conserved charged residues including Asp33 (in a conserved DT/SD motif), Arg57, and Arg132 (interacting with shikimate) are identified, guiding the development of novel inhibitors of shikimate kinase.

Inhibition of Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells by Phyllanthus urinaria extracts

AIM OF THE STUDY Helicobacter pylori is linked to a majority of peptic ulcers and to some types of gastric cancer, and its resistance to antibiotic treatment is now found worldwide. This study is aimed at evaluating the antimicrobial activity of Phyllanthus urinaria Linnea (Euphorbiaceae), chloroform (PUC) and methanol (PUM) extracts, and its eight isolates on H. pylori-infected human gastric epithelial AGS cells. MATERIALS AND METHODS The in vitro anti-bacterial activity of P. urinaria chloroform (PUC) and methanol (PUM) extracts, and its eight isolates were determined. Additional experiments were also performed to know the PUC and PUM ability to inhibit the H. pylori adhesion to and invasion of AGS cells, in addition to the effect of PUC on NF-kappaB activity as well as IL-8 synthesis during H. pylori infection of AGS cells. RESULTS The results revealed that crude extracts PUC and PUM showed potent antimicrobial activity against H. pylori than pure isolates. On the other hand, in vitroH. pylori-infection model revealed that the inhibition of bacterial adhesion and invasion to AGS cells has dramatically reduced by treatment of extract PUC, while PUM has the same moderate effect. Furthermore, H. pylori-induced nuclear factor (NF)-kappaB activation, and the subsequent release of interleukin (IL)-8 in AGS cells were also inhibited by the extract PUC. CONCLUSIONS These results open the possibility of considering P. urinaria a chemopreventive agent for peptic ulcer or gastric cancer, but this bioactivity should be confirmed in vivo in the future.

Crystal structure of Helicobacter pylori formamidase AmiF reveals a cysteine-glutamate-lysine catalytic triad

Helicobacter pylori AmiF formamidase that hydrolyzes formamide to produce formic acid and ammonia belongs to a member of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75Å resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an α-β-β-α four-layer architecture characteristic of the nitrilase superfamily. One exterior α layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight α-β-β-α-α-β-β-α dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30Å, respectively. These structures reveal a small formamide-binding pocket that includes Cys166, Glu60, and Lys133 catalytic residues, in which Cys166 acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl d-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently.

Expression of Trigonopsis variabilis D-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants

The D-amino acid oxidase cDNA gene (daao) of Trigonopsis variabilis was prepared by reverse transcriptase-polymerase chain reaction (PCR) and cloned into Escherichia coli expression vector, pTrc99A, under the control of tac promoter. Expression of daao gene significantly affected the growth and morphology of E. coli. The highest D-amino acid oxidase (DAAO) activity was 705 U (mg of protein)(-)(1), which was about 12-fold higher than that of D-alanine-induced T. variabilis. The DAAO protein exhibited activity on native-PAGE and had a M(r)value of 39.3 kDa. We also constructed an expression plasmid, pKm-DAAO, in which kanamycin instead of ampicillin was used as the selective marker. High-performance liquid chromatography (HPLC) analysis demonstrated that cephalosporin C could be converted to 7-glutarylcephalosporanic acid by cell-free extract of E. coli harboring pKm-DAAO. Four inactive DAAO mutants were obtained by error-prone PCR. Sequence analysis of these four DAAO mutants indicated the occurrence of mutations at Val-167, Pro-291, Pro-309, and Ala-343 residues. The His(6)-tagged DAAOs were expressed in E. coli and purified by nickel ion affinity chromatography. The results showed that all DAAO mutants lost their enzymatic activities and characteristic adsorption spectra for flavoenzyme. Based on the crystal structure of a homologous protein, pig DAAO, it is suggested that these four residues may play essential structural roles in DAAO conformation, thereby influencing DAAOs catalytic activity.