Sequence and apoptotic activity of VacA cytotoxin cloned from a Helicobacter pylori Thai clinical isolate

Abstract

The vacuolating cytotoxin VacA produced by Helicobacter pylori induces the formation of large cytoplasmic vacuoles in host gastric epithelial cells as well as a release of cytochrome C from mitochondria resulting in cell apoptosis. Considerable sequence diversity in VacA relating to different degrees of disease severity is observed with clinical samples from a multitude of geographic places. In this study we describe expression in Escherichia coli, purification to homogeneity and in vitro assay of its apoptotic activity of a VacA toxin from a H. pylori isolate of a Thai patient with gastrointestinal lymphoma. Sequencing revealed that the deduced amino acid sequence of the cloned Thai isolate VacA is similar to H. pylori s1/m2 type strains. The percent sequence similarity to the model strain 60190 was lower due to the presence of extra amino acids in the mid (m) region. The purified VacA toxin exhibited significant apoptotic activity on both T84 and MDCK epithelial cell lines, as revealed by DAPI staining, whereby the observed activity was significantly higher on MDCK cells. These findings could relate to a modulation of VacA activity on host cells in the Thai isolate-VacA toxin that may differ from those of the model strain.

Figure 1

Partial nucleotide sequence and deduced amino acid sequence in the m region of recombinant vacA toxin gene from Thai isolate (rVacA) aligned with sequences of H. pylori model strain 60190 (s1/m1) and strain 95-54 (s1/m2). (a) Nucleotide sequences identical in the 3 strains are in blue, and nucleotide sequences identical between 2 strains are shown in yellow. The black box shows the insertion in the middle region of the Thai isolate vacA gene. (b) Amino acid sequences identical in the 3 strains are in blue, and amino acid sequences identical between 2 strains are shown in yellow. The black box shows the insertion in the middle region of the Thai isolate vacA gene.

Figure 2

Purification and Western blot analysis of VacA. (a)  Purification of H. pylori VacA toxin by Ni²⁺affinity chromatography. The column was washed with 0.1 M Tris-HCl, pH 8.0, 0.3 M NaCl, 10 mM imidazole (peak a). VacA toxin was eluted with elution buffer (0.1 M Tris-HCl, pH 8.0, 0.3 M NaCl, 100 mM imidazole) (peak b). (b) SDS-PAGE (10% gel) analysis of H. pylori VacA toxin after IMAC purification. Lane M: broad range protein marker; lane 1: flow-through; lane 2: 10 mM imidazole wash; lane 3: 100 mM imidazole elution fraction of the protein. (c) Corresponding Western blot of H. pylori VacA toxin after IMAC purification. Western blot profile of the gel as seen in (a) using anti-VacA antibody on 10% SDS-PAGE. Lane M: broad range protein marker; lane 1: flow-through; lane 2: 10 mM imidazole wash; lane 3: 100 mM imidazole elution fraction of the protein.

Figure 3

DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with VacA (150 µg mL⁻¹) cells showing chromatin condensation and DNA fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL⁻¹) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.

Figure 4

Percentage of apoptotic cells as observed after DAPI staining in T84 and MDCK cells. A mean of six pictures from each sample with at least 200 cells was counted for apoptosis-positive cells and the mean percentage of apoptotic cells was compared with the untreated control. Error bars represent SEM.