Helicobacter pylorienter and survive within multivesicular vacuoles of epithelial cells

Abstract

SummaryAlthough intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell-associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco-2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time-lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half-life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori -containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA -mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.

Helicobacter pylori tissue tropism: mouse-colonizing strains can target different gastric niches

“…whether discrete physical compartments, or positions in gradients of metabolites or macromolecules, is not known. A compartment model is suggested by occasional findings of intracellular H. pylori (Allen, 2000;Amieva et al, 2002), if it is assumed that SS1 and X47 tend to occupy different cell types (one predominant in the gastric antrum, the other in the corpus). One attractive molecular gradient model was suggested by pH differences in the antrum vs corpus.…”

The NudA Protein in the Gastric Pathogen Helicobacter pylori Is an Ubiquitous and Constitutively Expressed Dinucleoside Polyphosphate Hydrolase

“…Therefore, it is not clear whether the NudA protein is involved in H. pylori invasion of gastric epithelial cells. However, considering the low frequency of invasion of H. pylori, the gentamicin protection assay might not be sensitive enough to monitor differences in invasion capacity since complete killing of extracellular bacteria is rarely obtained (20).…”

“…This Gram-negative, microaerophilic bacterium causes gastritis, gastric ulcer, and duodenal ulcer and is associated with gastric cancer (17,18). H. pylori is primarily an extracellular bacterium, but it has been observed intracellularly, although it is still unclear whether it is capable of multiplying and persisting within host cells (19,20). H. pylori is transformed from a dividing rod to a nonculturable coccoid form at unfavorable conditions, e.g.…”

Effect ofHelicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

“…4 Furthermore, VacA favors persistent infection through the inactivation of immune cells 5 and promoting H. pylori survival inside both macrophages and gastric epithelial cells, which may serve as potential intracellular reservoirs for the bacteria. [6][7][8][9][10] A hallmark of VacA intoxication of cultured epithelial cells is the formation of large intracellular vacuoles that can occupy a large fraction of the epinuclear region within the cytosol. 4 Vacuole biogenesis originates within late endosomal-lysosomal compartments, where VacA induces the fusion and/or swelling of membrane-bound compartments by a mechanism dependent on the recruitment of the small GTPase Rab7.…”

“…9,[11][12][13][14] While the exact role of VacAdependent vacuolation remains unclear, studies with infection of gastric epithelial cells revealed an increase in H. pylori intracellular survival associated with the presence of bacteria inside of both large and small VacA-dependent vacuoles. 6,9,15,16 The suitability of these vacuoles as an intracellular niche for H. pylori may be due, at least in part, to toxin-mediated alterations in the sorting of lysosomal hydrolases during vacuole biogenesis that yield nondegradative compartments. 9,12,17 Several of the earliest studies describing the action of VacA on cultured epithelial cell lines reported that broth culture supernatants from vacA + H. pylori strains induced the formation of intracellular vesicles that visibly stained with acridine orange, initially excluded the endocytic marker Lucifer yellow, and, appeared to contain cytoplasmic content.…”