Helicobacter pylori VacA toxin promotes bacterial intracellular survival in gastric epithelial cells

Abstract

Helicobacter pylori colonizes the gastric epithelium of at least 50% of the world's human population, playing a causative role in the development of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. Current evidence indicates that H. pylori can invade epithelial cells in the gastric mucosa. However, relatively little is known about the biology of H. pylori invasion and survival in host cells. Here, we analyze both the nature of and the mechanisms responsible for the formation of H. pylori's intracellular niche. We show that in AGS cells infected with H. pylori, bacterium-containing vacuoles originate through the fusion of late endocytic organelles. This process is mediated by the VacA-dependent retention of the small GTPase Rab7. In addition, functional interactions between Rab7 and its downstream effector, Rab-interacting lysosomal protein (RILP), are necessary for the formation of the bacterial compartment since expression of mutant forms of RILP or Rab7 that fail to bind each other impaired the formation of this unique bacterial niche. Moreover, the VacA-mediated sequestration of active Rab7 disrupts the full maturation of vacuoles as assessed by the lack of both colocalization with cathepsin D and degradation of internalized cargo in the H. pylori-containing vacuole. Based on these findings, we propose that the VacA-dependent isolation of the H. pylori-containing vacuole from bactericidal components of the lysosomal pathway promotes bacterial survival and contributes to the persistence of infection.

FIG. 1.

VacA toxin is involved in the formation of the H. pylori intracellular niche and promotes bacterial survival. A DIC micrograph of uninfected control AGS cells is shown in panel A. AGS cells after 24 h of invasion with wild-type H. pylori is shown in panel B. The inset in panel B shows details of the H. pylori-containing vacuoles. Arrows point to intracellular bacteria inside the vacuolar compartment. Panel C shows AGS cells after 24 h of invasion with H. pylori vacA mutant strain. After 10 h of infection with the H. pylori vacA mutant strain, AGS cells were treated with wild-type H. pylori conditioned culture medium for 24 h (D). The inset in panel D shows details of the H. pylori-containing vacuoles, and the arrows point to intracellular bacteria inside the vacuolar compartment. AGS cells were infected with wild-type or vacA mutant H. pylori strains under gentamicin assay conditions (see Materials and Methods). At the indicated invasion times, intracellular H. pylori cells were retrieved from AGS cells utilizing 1% saponin in PBS buffer and plated on brucella agar. Intracellular survival (CFU) of wild-type (triangles) and vacA mutant (squares) H. pylori strains is shown in panel E. (CFU values corresponded to means ± standard error from a single representative experiment performed in triplicate. These results were reproduced on four separate occasions). AGS cells were infected with wild-type [VacA (+)] or VacA-negative mutant [VacA (−)] H. pylori. Three hours after infection with the vacA mutant strains, AGS cells were treated with wild-type H. pylori CCM or PVT, and intracellular survival (CFU) of the bacteria was determined using the gentamicin protection assay at the 36-h invasion time point (F). (CFU values corresponded to means ± standard error from a single representative experiment performed in triplicate. These results were reproduced on four separate occasions.) *, P = 0.0001; **, P = 0.0003.

FIG. 2.

Helicobacter pylori's intracellular compartment acquires late endosomal and lysosomal markers. Panels A and B show the distribution of GFP-Rab7 (green) and Lamp-1 (blue) protein for control (A) and wild-type H. pylori-invaded cells (B). Details of the vacuolar compartment showing Lamp1 and Rab7 recruitment are presented in panels C and D, respectively. Panel E shows the distribution of GFP-Rab7 (green) and Lamp-1 (blue) for AGS cells invaded by an H. pylori vacA mutant strain. The inset in panel E shows in detail the morphology of the intracellular compartment of the vacA mutant bacteria. The recruitment of Lamp1 and Rab7 to the bacterial compartment is shown in detail in panels F and G, respectively. Panels H to J show the distribution of GFP-CD63 (green) for uninfected AGS cells (H) and AGS cells infected with wild-type (I) or VacA mutant (J) H. pylori, respectively. The insets in panels I and J show details of the bacterial niches. All of the microphotographs were taken with a spinning disk confocal microscope with a ×100 oil objective. The scale bar in panel I is equivalent to 3 μm. Immunolabeled bacteria are shown in red. For all experiments, the invasion time was 24 h.

FIG. 3.

Assessing the pH of H. pylori's intracellular compartment. H. pylori-containing vacuoles loaded with the pH-sensitive fluorescent dye Oregon green 514 are shown in panels A (DIC of invaded cells) and B. The result of a typical pH determination assay representative of three independent experiments is shown in panel C. Oregon green fluorescent ratio (excitation at 510/450 nm detected at 530) were measured in 10 different bacterial compartments distributed in different cells along the microscope optical field (black lines) and plotted as a function of the pH equilibration time. An average of the ratio for the fluorescent background of the field was obtained in a cell-free area (dashed line). The section of the curve between two arrowheads indicates the fluorescent ratio for H. pylori vacuoles at the resting state in isosmotic Na solution. The arrow indicates the time when nigericin isosmotic K⁺ solution was added and the onset for the pH calibration of the bacterial compartments. The pH corresponding to each equilibration time is indicated above the curves. The fluorescence ratio/pH titration curve is shown in figures in the supplemental material.

FIG. 4.

VacA toxin prevents the sorting of cathepsin D to H. pylori-containing vacuoles in invaded AGS cells. Panel A shows GFP-Rab7 (green)-transfected AGS cells invaded with VacA mutant H. pylori bacteria. Thirty-two hours after infection, cells were fixed, permeabilized, and immunolabeled for cathepsin D (red). H. pylori cells were labeled with DAPI (4′,6′-diamidino-2-phenylindole) (blue). Panels B and C show in detail the bacterial intracellular niche from the area enclosed in panel A. Panel D shows GFP-Rab7 (green)-transfected AGS cells invaded with wild-type H. pylori. Cells were fixed, permeabilized, and immunolabeled for cathepsin D (red). H. pylori was labeled with DAPI (blue). Panels E and F show in detail the bacterial intracellular niche from the area enclosed in panel D. Ten hours after infection with vacA mutant H. pylori, GFP-Rab7-expressing AGS cells (green) were incubated for 20 h with wild-type conditioned growth medium (CCM) (G). Cells were fixed, permeabilized, and immunolabeled for cathepsin D (red). H. pylori was labeled with DAPI (blue). An uninfected AGS cell expressing GFP-Rab7 (green) and immunolabeled for cathepsin D (red) is shown in panel J. The average number of cathepsin D-positive bacteria in either large or small compartments per cell is shown in panel K. Intracellular wild-type H. pylori cells were scored according to their association with cathepsin D and the size of the vacuolar compartment. Small compartments were arbitrarily defined as vacuoles that closely surround the bacteria (arrowheads), while large compartments clearly surpassed the bacterial volume (arrows). The number of bacteria was assessed from 15 infected cells utilizing z-stack micrograph slices obtained each 0.25 μm by confocal microscopy. *, P = 0.03. The white bars in the micrographs indicate 10-μm scale. Original magnification, ×1,000.

FIG. 5.

H. pylori invasion of AGS cells inhibits lysosomal protease activity in a VacA-dependent manner. GFP-Rab7 (green)-expressing cells were infected with vacA mutant bacteria (A and B) or wild-type (C and D) H. pylori. Following 24 h of infection, the cells were incubated with the chromogenic protease substrate DQ red-BSA (red) for a 3- to 4-h period. After this time, the cells were fixed and the bacteria were labeled with DAPI (4′,6′-diamidino-2-phenylindole). Panel E shows GFP-Rab7 (green)-expressing cells infected with wild-type H. pylori. Twenty-four hours later, the cells were incubated with a fluid-phase fluorescence marker, rhodamine dextran (0.5 mg/ml) (red), for a 3- to 4-h period, extensively washed, and then examined by confocal microscopy.

FIG. 6.

H. pylori causes fusion of phagolysosome compartments in CHOIIa cells. Panel A shows a DIC micrograph of 3-μm latex bead-containing phagolysosomes in CHOIIa cells. The exposure of CHOIIa cells to wild-type but not vacA mutant H. pylori caused the fusion of latex bead-containing phagolysosomes. (B and C) DIC images of latex bead-containing phagolysosomes in CHOIIa cells expressing GFP-Rab7 (green label in left corner of the panels) invaded with wild-type (B) or vacA mutant (C) H. pylori strains.

FIG. 7.

H. pylori invasion causes fusion of phagolysosome and bacterial compartments in FcIIa-expressing AGS cells. Panel A shows a fluorescent confocal micrograph of GFP-Rab7-positive phagosomes in FcIIa-expressing AGS cells, and panel B shows the merged image with the bright-field micrograph showing single 3-μm latex beads contained in each of the compartments. The exposure of AGS cells to wild-type H. pylori for a 12-h period caused the fusion of latex bead-containing phagolysosomes to form large GFP-Rab7-positive vacuoles (C), which contain multiple latex beads as shown in panel D. The exposure of AGS cells to vacA mutant H. pylori for a 12-h period did not alter the morphology of Rab7-positive engineered phagolysosomes (E and F). A DIC micrograph of an AGS cell infected with H. pylori containing multiple 3-μm latex beads in single large vacuoles is shown in panel G. Panel H shows details of the vacuole indicated in panel G. The arrows in panel H show motile intracellular H. pylori cells sharing the same vacuolar compartment with latex beads.

FIG. 8.

Rab7 and its effector RILP are necessary for the morphogenesis of H. pylori-containing vacuoles. Wild-type GFP-Rab7 (green) (A), but not GFP-Rab7 mutant F45A (green) (B), was recruited to H. pylori (blue)-containing vacuoles in AGS cells following 24 h of invasion (A and B, respectively). The Rab7 effector protein GFP-RILP (red) was recruited to H. pylori (blue)-containing vacuoles in AGS cells after 24 h of infection (C). Panel D shows that the expression of the GFP-Rab7 mutant, V180A (green), in AGS cells inhibited the formation of the large intracellular bacterial compartment after 24 h of infection. Panel E depicts inhibition of the large bacterium-containing compartment when the myc-tagged RILP mutant I251A is coexpressed with GFP-Rab7 (green) in AGS cells. Coexpression of myc-tagged Rab7 and mutant RILP C33 GFP (red) inhibits the morphogenesis of H. pylori's (blue) large compartment (F) at 24 h of infection. Panel G shows the distribution of Rab34 GFP (green) in AGS cells after 24 h of infection with H. pylori (blue). Panel H shows quantitation of bacterium-mediated vacuolation in H. pylori-infected cells transfected with various mutant forms of Rab7 or RILP. One hundred cells in three independent experiments were scored for vacuole formation 24 h after bacterial invasion. Data are mean ± standard error. H. pylori cells were stained with the nucleic acid dye DAPI (4′,6′-diamidino-2-phenylindole) (blue).

FIG. 9.

VacA-positive H. pylori cells recruit and retain active Rab7 to their intracellular compartment. AGS cells were transfected with GFP-Rab7 and then infected with H. pylori for 24 h. GFP-Rab7 mobility was then estimated by FRAP. Panel A shows a representative quantitation and nonlinear regression fitted curve of the fluorescent recovery of Rab7 in endosomes from uninfected cells (blue line and triangles) and the H. pylori compartment (red line and squares). The summary of the fractional recovery of Rab7 in endosomes (light gray bar) and bacterial vacuoles (dark gray bar) from invaded cells is shown in panel B. Data are mean ± standard error of 10 cells.