Analysis of Helicobacter pylori VacA-containing vacuoles and VacA intracellular trafficking

The human pathogen Helicobacter pylori colonizes half of the global
population. Residing at the stomach epithelium, it contributes to
the development of diseases like gastritis, duodenal and gastric
ulcers, and gastric cancer. It has evolved a range of mechanisms to
aid in colonization and persistence, manipulating the host immune
response to avoid clearance. A major factor in this is the secreted
vacuolating cytotoxin VacA which has a variety of effects on host
cells. VacA is endocytosed and forms anion-selective channels in
the endosome membrane, causing the compartment to swell. The
resulting VacA-containing vacuoles (VCVs) can take up most of the
cellular cytoplasm. Even though vacuolation is VacA's most
prominent and namesake effect, the purpose of the vacuoles is still
unknown. VacA exerts influence on the host immune response in
various ways, both pro- and anti- inflammatorily. Most importantly,
it disrupts calcium signaling in T-lymphocytes, inhibiting T-cell
activation and proliferation and thereby suppressing the host
immune response. Furthermore, VacA is transported to mitochondria,
where it activates the mitochondrial apoptosis pathway. Within the
cell, VacA has only been shown to localize to endocytic
compartments/VCVs and mitochondria. Considering its diverse
effects, however, the existence of other cellular sites of action
seems plausible. In this study, the VCV proteome was
comprehensively analyzed for the first time in order to investigate
VCV function. To this end, three different strategies for VCV
purification from T-cells were devised and tested. Eventually, VCVs
were successfully isolated via immunomagnetic separation, using a
VacA-specific primary antibody and a secondary antibody coupled to
magnetic beads. The purified vacuoles were then measured by mass
spectrometry, revealing not only proteins of the endocytic system,
but also proteins usually localized in other cellular compartments.
This apparent recruitment of proteins involved in all kinds of
cellular pathways indicates a central function of VCVs in VacA
intoxication effects. In a global evaluation, the VCV proteome
exhibited an enrichment of proteins implicated in immune response,
cell death, and cellular signaling; all of these are processes that
VacA is known to influence. One of the individual proteins
contained in the sample was STIM1, a calcium sensor normally
residing in the endoplasmic reticulum (ER) that is important in
store- operated calcium entry (SOCE). This corroborates the
findings of a concurrent report, in which VacA severely influenced
SOCE and colocalized with STIM1. A direct interaction of STIM1 with
VacA was examined in a pull-down assay, but could be neither shown
nor excluded. Immunofluorescence experiments conducted in HeLa
cells confirmed the presence of VacA in the ER and also found it to
traffic to the Golgi apparatus, identifying these two cellular
compartments as novel VacA target structures. The exact route of
VacA transport remains unclear, but the involvement of both the ER
and the Golgi suggests the possibility of retrograde trafficking,
analogous to other bacterial toxins like shiga and cholera toxins.
In summary, the elucidation of the VCV proteome and the discovery
of the ER and the Golgi apparatus as VacA target structures have
generated intriguing starting points for future studies. The
detection of many proteins implicated in VacA intoxication effects
in the VCV proteome leads to the proposal of VCVs as signaling hubs
that may coordinate the complex meshwork of VacA effects. Further
investigation of individual proteins is expected to help greatly in
illuminating this matter.