Effects of Helicobacter pylori Vacuolating Cytotoxin A on intracellular calcium signalling in T-lymphocytes

More than 50% of the world's population harbor Helicobacter pylori
in their stomach mucosa. The chronic gastric infection is
associated with several diseases including peptic ulcer disease and
gastric carcinoma. One of the most thoroughly studied virulence
factors produced by H. pylori is the Vacuolating Cytotoxin A
(VacA). All isolated H. pylori strains possess the vacA gene,
although significant sequence diversity was noticed in vacA genes
across H. pylori isolates. VacA protein is produced and secreted as
an 88 kD mature toxin. The protein binds to the host cells and is
internalized. Inside the host cells, it causes “vacuole”-like
membrane vesicles in the cytoplasm of gastric epithelial cells.
Besides vacuolation, VacA exerts various other effects on target
cells. VacA also forms membrane-embedded pores at the
inner-mitochondrial membrane, resulting in mitochondrial
dysfunction by cytochrome c release and apoptosis induction. VacA
suppresses nuclear translocation of nuclear factor of activated
T-cells (NFAT) resulting in down regulation of interleukin-2 (IL2)
gene transcription to efficiently block proliferation of T-cells.
The aim of this work was to understand the effects of VacA on
intracellular calcium signalling in T-lymphocytes by considering
the fact that VacA inhibits the Ca2+-calmodulin-dependent
phosphatase calcineurin and induces cell cycle arrest. However, the
exact mechanism how VacA exerts this response in T-cells is not
known. Therefore, in this thesis various cell lines were used to
study the effects of VacA on calcium influx. Calcium influx was
found to be affected in the presence of VacA protein in the human
Jurkat E6.1 T-cell line and primary human CD4+ T-cells activated by
phorbol myristate acetate (PMA). Once inside T-cells, it could be
shown that VacA suppresses the increase of the cytosolic free
calcium concentration after stimulation by the calcium ionophore
ionomycin and thapsigargin. Ionomycin forms pores in the
cytoplasmic membrane, whereas thapsigargin blocks the
sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and
thereby causes depletion of the endoplasmic reticulum (ER) calcium
store. In contrast, a VacA mutant, which was constructed by
deletion of the hydrophobic region (amino acids 6-27), was unable
to induce vacuolation activity and to block Ca2+ influx. A major
result of this work was to demonstrate that one of the main
components of store operated calcium entry (SOCE), the ER localized
calcium sensor protein STIM1, is a target of VacA. Using
co-localization studies and yeast two-hybrid (YTH) assays, it was
found that VacA localizes to the lumen of the ER where it binds to
the cEF-hand domain of STIM1. Furthermore, these data show that
VacA strongly reduced the movements of the STIM1 towards the plasma
membrane localized calcium channel ORAI1 after Ca2+ store depletion
by thapsigargin. A YTH screen identified cEF-hand domain of STIM1
as the target of VacA to inhibit calcium influx. The results
obtained in this work showing involvement of VacA in the modulation
of intracellular calcium signalling will provide new insights that
are required to understand how VacA inhibits T-cell proliferation
and signalling.