Specific entry of Helicobacter pylori into cultured gastric epithelial cells via a zipper-like mechanism

Abstract

Although Helicobacter pylori has generally been considered an extracellular pathogen, a number of in vitro infection experiments and biopsy examinations have shown that it is capable of occasionally entering mammalian host cells. Here, we characterized this entry process by using AGS cells as a host cell model. In gentamicin protection-invasion assays, the number of H. pylori colonies recovered was lower than that for Salmonella enterica serovar Typhimurium X22, Escherichia coli expressing InvA, and Yersinia enterocolitica YO:9 grown at 25 degrees C but higher than that for Neisseria gonorrhoeae VP1 and Y. enterocolitica YO:9 grown at 37 degrees C. At the ultrastructural level, the entry process was observed to occur via a zipper-like mechanism. Internalized H. pylori was bound in tight LAMP-1-containing vacuoles in close association with condensed filamentous actin and tyrosine phosphorylation signals. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, and calphostin C, an inhibitor of protein kinase C, both inhibited the entry of H. pylori in a sensitive and dose-dependent manner; however, the level of entry was enhanced by sodium vanadate, an inhibitor of tyrosine phosphatases and ATPases. Furthermore, the cytokine tumor necrosis factor alpha antagonized the entry of H. pylori into AGS cells. Collectively, these results demonstrate that the entry of H. pylori into AGS cells occurs via a zipper-like mechanism which involves various host signal transduction events.

FIG. 1.

Strain-to-strain variation in the frequency of host cell entry. Confluent AGS cell monolayers were infected with the various strains of H. pylori at an MOI of 100 for 3 h at 37°C. Intracellular (black bars) and adherent (white bars) bacteria were detected by IF labeling. AGS cells were stained for F actin. Quantitation of intracellular and adherent H. pylori was performed by CLSM. Data were obtained in triplicate from at least two independent experiments. Error bars indicate standard deviations.

FIG. 2.

Kinetics of entry of H. pylori into AGS cells. (A) Time course of infection during the first 3 h of infection of AGS cells with H. pylori 26695 at an MOI of 100. Intracellular (black bars) and adherent (white bars) H. pylori 26695 bacteria were quantitated as described in the legend to Fig. 1. (B) Recovery of viable intracellular bacteria during 24 h of infection. The number of CFU of viable cell-associated adherent (white bars) and intracellular (black bars) H. pylori at the various time points was determined by the gentamicin protection assay. Data were obtained in triplicate from at least three independent experiments and are presented as the number of viable cell-associated bacteria or the number of viable intracellular bacteria per well of AGS cells. Error bars indicate standard deviations.

FIG. 3.

SEM and TEM analyses of H. pylori 26695 adherence to and entry into AGS cells. AGS cells were infected with H. pylori 26695 at an MOI of 100 for 12 h at 37°C. Samples obtained at various times postinfection were analyzed by SEM and TEM. H. pylori adhered to AGS cells by intimate contact with the host cell microvilli (A and B, arrows) and pseudopods (C, arrows). Fibrillar structures connecting adherent H. pylori to AGS cells were occasionally seen (E, arrows). Features of zipper-like engulfment (D, F, G, and H, arrows) were observed at 1 to 3 h postinfection, whereas adherence to the host cell periphery was prominent by 12 h postinfection (I, arrows). Bars, 1.5 μm.

FIG. 4.

Transmission electron micrograph showing the intracellular location of H. pylori 26695 in AGS cells 3 h postinfection. AGS cells were infected with H. pylori 26695 at an MOI of 100 for 1.5 h at 37°C. Samples were fixed and subsequently analyzed by TEM. The phagosomal membrane is indicated by arrows.

FIG. 5.

Confocal laser scanning micrograph showing the localization of H. pylori in LAMP-1-containing phagosomes 3 h postinfection. AGS cell monolayers were infected with H. pylori 26695 at an MOI of 100 for 3 h at 37°C. Samples were then fixed and subjected to triple IF labeling. Total H. pylori was stained blue (C). Extracellular H. pylori was stained green (A) and appeared light blue in the overlay (D, arrowheads). The human LAMP-1 protein was stained red (B and D, small arrows). Intracellular H. pylori which colocalized with the LAMP-1-containing phagosomes appeared magenta (D, large arrows). Bar, 10 μm.

FIG. 6.

CLSM analysis of the spatial relationship among intracellular H. pylori, host actin cytoskeleton, and protein phosphotyrosine signals. AGS cells were infected with H. pylori 26695 at an MOI of 100 for 3 h at 37°C, washed, and fixed with PFA. Specimens were stained for F actin (A, E, J, N, and R), for phosphotyrosine (B, F, K, O, and S), and for H. pylori (C, G, L, P, and T). Images were obtained in various focal planes (panels in rows) by scanning from the apex to the basal side of the host cell (panels in columns, from top to bottom). The proximity between bacteria and phosphotyrosine signals is indicated by arrowheads in the overlay of the three channels (D, H, M, Q, and U) and in enlargement in inset I, whereas the close association of bacteria with F actin can be seen. The orange color indicates colocalization of F actin with phosphotyrosine (M and Q, arrows). Bar, 10 μm.

FIG. 7.

Effects of preinfection of Y. enterocolitica YO:9 grown at either 37or 25°C on the level of H. pylori entry into AGS cells. AGS cells were first infected with Y. enterocolitica YO:9 grown at 37 or 25 ^oC for 1 h at an MOI of 50 and then infected with H. pylori 26695 at an MOI of 50 for 3 h. Intracellular (black bars) and adherent (white bars) bacteria were detected by IF labeling as described in the text. Intracellular and adherent H. pylori bacteria were quantitated by CLSM. Error bars indicate standard deviations.

FIG. 8.

Effect of wortmannin, calphostin C, sodium ortho-vanadate, or TNF-α on the level of H. pylori entry into AGS cells. Wortmannin (A), calphostin C (B), sodium ortho-vanadate (C), or TNF-α (D) was added to AGS cultures at the appropriate concentration, and the cultures were incubated at 37°C for 15 min. H. pylori 26695 was then added to the cell cultures at an MOI of 100. Infection was carried out for 3 h at 37°C in the presence of the inhibitor, after which samples were washed and fixed with PFA. Staining for intracellular and extracellular bacteria and host cells was performed as described in the text. Intracellular (•) and adherent (○) H. pylori 26695 bacteria were quantitated by CLSM. Data obtained were consistent among the staining strategies. Error bars indicate standard deviations.