Journal: Infection and Immunity

Article Title: The Intermediate Region of Helicobacter pylori VacA Is a Determinant of Toxin Potency in a Jurkat T Cell Assay

doi: 10.1128/IAI.00052-12

Figure Lengend Snippet: Amino acid sequences of the VacA i-region in different H. pylori strains. The secreted p88 VacA protein (comprising 821 amino acids in H. pylori strain 60190) contains two domains, designated p33 and p55. Amino acid sequence variation in VacA proteins from different H. pylori strains is especially prominent in three regions, which are designated s-, i-, and m-regions. The i-region and a portion of the s-region are localized within the p33 domain, and the m-region is localized within the p55 domain. The lower portion of this figure shows the sequences of VacA i-regions from strain 60190 (GenBank no. U05676), Tx30a (GenBank no. U29401), and X47. The figure illustrates the sequence of the secreted VacA protein of H. pylori strain 60190 (amino acids G120 to L236) and corresponding regions of VacA in two other wild-type strains (Tx30a and X47). Clusters of amino acid polymorphisms defined as clusters A, B, and C are indicated. Asterisks (*) indicate amino acids that are identical in all three sequences. The sequence of VacA from strain 60190 is designated a prototype for the i1 region, and the sequence of VacA from strain Tx30a is designated a prototype for the i2 region. VacA from strain X47 contains a type i2 sequence in cluster C, and cluster B is chimeric.

Article Snippet: H. pylori liquid cultures were grown in brucella broth supplemented with either activated charcoal or 5% fetal bovine serum (FBS) ( 11 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain/plasmid Relevant characteristics Reference Strains 60190 Wild type (ATCC 49503); vacA s1/i1/m1 40 60190 Δ rdxA Same as 60190 except HP0954 ( rdxA ) gene deleted; metronidazole resistant This study 60190 vacA :: cat rdxA Same as 60190 Δ rdxA except cat cassette and rdxA inserted in vacA ; chloramphenicol resistant and metronidazole sensitive; expression of VacA is disrupted This study 60190 i2B Same as 60190 Δ rdxA except vacA cluster B changed to i2 This study 60190 i2C Same as 60190 Δ rdxA except vacA cluster C changed to i2 This study 60190 i2BC Same as 60190 Δ rdxA except vacA clusters B and C changed to i2 This study 60190 i1/i2C Same as 60190 Δ rdxA except vacA cluster C has 4 amino acids changed to i2 This study X47 Wild type; vacA s1/m2, chimeric i-region 32 X47 Δ rdxA Same as X47 except HP0954 ( rdxA ) gene deleted; metronidazole resistant This study X47 vacA :: cat rdxA Same as X47 Δ rdxA except cat cassette and rdxA inserted in vacA ; chloramphenicol resistant and metronidazole sensitive; expression of VacA is disrupted This study X47 i1C Same as X47 Δ rdxA except vacA cluster C changed to i1 This study Plasmids pMM672 Allows deletion of rdxA in H. pylori strains 41 pCGR1 Contains cat-rdxA cassette in StuI site; derived from pA178 plasmid This study pCGR2 Contains cat-rdxA cassette in EcoRV site from X47 vacA This study pCGR3 60910 cluster B changed from i1 to i2 by inverse PCR using primers B1F and B1R This study pCGR4 60190 cluster C changed from i1 to i2 by inverse PCR using primers C1F and C1R This study pCGR5 60190 clusters B and C changed from i1 to i2 by inverse PCR using primers C1F and C1R and pCGR3 as the template This study pCGR6 A portion of 60190 cluster C changed from i1 to i2 by inverse PCR using primers C2F and C2R This study pCGR7 X47 cluster C changed from i2 to i1 by inverse PCR using primers C3F and C3R This study p55 Expresses VacA p55 27 p33 Expresses VacA p33 31 p33 i2 Expresses p33 i2 This study Open in a separate window H. pylori strains and plasmids

Techniques: Sequencing

Journal: Infection and Immunity

Article Title: The Intermediate Region of Helicobacter pylori VacA Is a Determinant of Toxin Potency in a Jurkat T Cell Assay

doi: 10.1128/IAI.00052-12

Figure Lengend Snippet: H. pylori strains and plasmids

Article Snippet: H. pylori liquid cultures were grown in brucella broth supplemented with either activated charcoal or 5% fetal bovine serum (FBS) ( 11 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain/plasmid Relevant characteristics Reference Strains 60190 Wild type (ATCC 49503); vacA s1/i1/m1 40 60190 Δ rdxA Same as 60190 except HP0954 ( rdxA ) gene deleted; metronidazole resistant This study 60190 vacA :: cat rdxA Same as 60190 Δ rdxA except cat cassette and rdxA inserted in vacA ; chloramphenicol resistant and metronidazole sensitive; expression of VacA is disrupted This study 60190 i2B Same as 60190 Δ rdxA except vacA cluster B changed to i2 This study 60190 i2C Same as 60190 Δ rdxA except vacA cluster C changed to i2 This study 60190 i2BC Same as 60190 Δ rdxA except vacA clusters B and C changed to i2 This study 60190 i1/i2C Same as 60190 Δ rdxA except vacA cluster C has 4 amino acids changed to i2 This study X47 Wild type; vacA s1/m2, chimeric i-region 32 X47 Δ rdxA Same as X47 except HP0954 ( rdxA ) gene deleted; metronidazole resistant This study X47 vacA :: cat rdxA Same as X47 Δ rdxA except cat cassette and rdxA inserted in vacA ; chloramphenicol resistant and metronidazole sensitive; expression of VacA is disrupted This study X47 i1C Same as X47 Δ rdxA except vacA cluster C changed to i1 This study Plasmids pMM672 Allows deletion of rdxA in H. pylori strains 41 pCGR1 Contains cat-rdxA cassette in StuI site; derived from pA178 plasmid This study pCGR2 Contains cat-rdxA cassette in EcoRV site from X47 vacA This study pCGR3 60910 cluster B changed from i1 to i2 by inverse PCR using primers B1F and B1R This study pCGR4 60190 cluster C changed from i1 to i2 by inverse PCR using primers C1F and C1R This study pCGR5 60190 clusters B and C changed from i1 to i2 by inverse PCR using primers C1F and C1R and pCGR3 as the template This study pCGR6 A portion of 60190 cluster C changed from i1 to i2 by inverse PCR using primers C2F and C2R This study pCGR7 X47 cluster C changed from i2 to i1 by inverse PCR using primers C3F and C3R This study p55 Expresses VacA p55 27 p33 Expresses VacA p33 31 p33 i2 Expresses p33 i2 This study Open in a separate window H. pylori strains and plasmids

Techniques: Expressing, Derivative Assay, Plasmid Preparation, Inverse PCR

Journal:

Article Title: A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1 Helicobacter pylori VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation

doi: 10.1128/JB.183.22.6499-6508.2001

Figure Lengend Snippet: Sequence diversity in the amino termini of s1- and s2-type toxins. (A) Amino-terminal amino acid sequences of type s1 (from H. pylori strain 60190) and s2 (from H. pylori strains Tx30a and VM083) VacA toxins. The arrows indicate the sites at which the amino-terminal signal sequences are cleaved. The 12-amino-acid amino-terminal extension found in s2-type toxins is underlined. (B) Predicted hydrophilicity of VacA toxins produced by H. pylori strains 60190, Tx30a, and VM083. The analysis is limited to the amino-terminal portion of each mature toxin. The amino acid numbering is based on the initiating methionine of the protoxins as amino acid 1. The 12-amino-acid amino-terminal extension found in s2 toxins is predicted to increase the hydrophilicity of the VacA amino terminus.

Article Snippet: H. pylori strains 60190 (ATCC 49503) and Tx30a (ATCC 51932) contain prototypes for two highly divergent families of vacA alleles (designated type s1/m1 and type s2/m2, respectively) ( 1 , 10 ).

Techniques: Sequencing, Produced

Journal:

Article Title: A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1 Helicobacter pylori VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation

doi: 10.1128/JB.183.22.6499-6508.2001

Figure Lengend Snippet: Vacuolating activities of VacA toxins from strains 60190 (s1/m1) and VM083 (s2/m1). Purified, acid-activated VacA preparations from H. pylori strains 60190 (●) and VM083 (○) were incubated with HeLa cells in MEM containing 10 mM ammonium chloride for 16 h at 37°C. Vacuolating activity was quantified using a neutral red uptake assay (9). The results represent the mean (± standard deviation) net absorbance at 540 nm from triplicate samples. VacA from strain 60190 induced cell vacuolation, whereas the chimeric s2/m1 VacA from strain VM083 did not.

Article Snippet: H. pylori strains 60190 (ATCC 49503) and Tx30a (ATCC 51932) contain prototypes for two highly divergent families of vacA alleles (designated type s1/m1 and type s2/m2, respectively) ( 1 , 10 ).

Techniques: Purification, Incubation, Activity Assay, Standard Deviation