Invasion and multiplication of Helicobacter pylori in gastric epithelial cells and implications for antibiotic resistance

Abstract

Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that infects more than 50% of the human population and can cause gastritis, peptic ulcer, or gastric malignancies. It is generally viewed as an extracellular microorganism. In a gentamicin protection assay on AGS or MKN45 cells, H. pylori could invade the epithelial cells and multiply within double-layer vesicles either on the plasma membrane or in the cytoplasm. A 5-fold increase in the number of bacteria was recultured from the infected cells at 12 h, compared with the number of invading cells at 2.5 h postinfection. The autophagic vesicles induced by H. pylori are the sites of replication and also of the degradation of the replicating bacteria after fusion with lysosomes. Many H. pylori bacteria in coccoid form associated with the plasma membrane can be released into culture. Only cell-penetrating antibiotics can enhance the intracellular killing of the replicating bacteria. The multiplication of H. pylori within cells provides a niche for its resistance to antibacterial therapy and has a significant impact on its biological life cycle.

FIG. 1.

Multiplication of H. pylori in epithelial cells. (a) H. pylori infection of AGS or MKN45 cells. (b) Infection of AGS cells by various strains of H. pylori. (c) Infection of AGS cells by cagA, babA, and vacA mutants of H. pylori. Epithelial cell lines AGS or MKN45 were infected with HP238 or various strains or mutants at an MOI of 50 for 1 h. Gentamicin was present throughout the culture period. Recovery of viable cell-associated H. pylori (CFU) was determined by a gentamicin protection assay at various times postinfection. *, P < 0.05. Each experiment was repeated at least three times with the same results. Values are the means ± standard deviations from one representative experiment.

FIG. 2.

Localization of replicating H. pylori in infected AGS cells. (a) Immunofluorescent staining of H. pylori in AGS cells. AGS cells were infected with HP238 at an MOI of 50 for 1 h. The infected cells were collected, fixed, and stained with anti-H. pylori antibody (red) and DAPI (blue) at various time points postinfection. The samples were observed with confocal or phase-contrast microscopy. The cell boundary is shown by dashed line. The plasma membrane-associated H. pylori bacteria are indicated by arrows. (b) Internalization of H. pylori on EEA1⁺ endosomes. AGS cells were infected with HP238 at an MOI of 20 for 1 h. The infected cells were collected, fixed, and stained with anti-H. pylori antibody (red) and anti-EEA1 antibody (green). The samples were observed with confocal microscopy. Arrows indicate the colocalization of H. pylori with the EEA1 early endosome marker. Arrowheads indicate no colocalization. (c) H. pylori on LAMP1⁺ lysosomes. As stated for panel b, the infected cells were stained with anti-H. pylori antibody (red) and anti-LAMP1 antibody (green). Arrows indicate the colocalization of H. pylori with the LAMP1 lysosome marker. Arrowheads indicate no colocalization. (d) Flow cytometric staining of H. pylori in AGS cells. AGS cells were infected with HP238 at an MOI of 25 for 1 h. Gentamicin (Gm; 150 μg/ml) was added to kill the extracellular bacteria. The infected cells were then cultured in gentamicin-containing medium (25 μg/ml) or gentamicin-free medium beginning at 5.5 h. At various time points, the infected cells were collected and stained with anti-H. pylori antibody without fixation. The mean fluorescence intensity of plasma membrane-associated-H. pylori in an arbitrary unit is shown to indicate the increased amount of anti-H. pylori staining. (e) Release of H. pylori into the culture supernatant. As stated for panel d, the supernatant from the infected AGS cells was collected for the recovery of the viable H. pylori (CFU). *, P < 0.05. Each experiment was repeated at least three times with the same results. Values are the means ± standard deviations from one representative experiment.

FIG. 3.

Autophagy induction by H. pylori in AGS cells. (a) H. pylori resides and replicates in double-layer vesicles. AGS cells were infected with HP238 at an MOI of 50 for 1 h. The infected cells were collected, fixed, and observed with electron microscopy at various time points postinfection. The fate of H. pylori postinfection is shown in a time sequence, as indicated on the figure. N, nucleus; MV, multiple-layered vesicle. Black arrows indicate double-layer membranes, whereas white arrows indicate the replicating H. pylori bacteria. White arrowheads indicate the multiple vacuoles, whereas black arrowheads indicate the coccoid forms of H. pylori. (b) The ratio of spiral to coccoid forms on the infected cells at various time points postinfection is shown. (c) LC3 punctate formation colocalized with H. pylori. The arrows indicate the colocalization of LC3 and H. pylori-containing vesicles. (d) Autophagic flux was induced post-H. pylori infection. LC3-II conversion, BNIP3, and p62 were induced postinfection. Each experiment was repeated at least two times with the same result.

FIG. 4.

The effect of antibiotic on the multiplication of H. pylori in AGS cells. AGS cells were infected with HP238 at an MOI of 50 for 1 h. Gentamicin (150 μg/ml) was added to kill the extracellular bacteria. The infected cells were then cultured in gentamicin-containing medium (25 μg/ml) or another antibiotic-containing medium beginning at 5.5 h. At various time points, the infected cells were collected and lysed for recovery of the viable cell-associated H. pylori (CFU). For HP238, the MICs of clarithromycin, metronidazole, and amoxicillin are 0.06 μg/ml, 32 μg/ml, and 0.06 μg/ml, respectively. For ATCC 43504, the MIC of clarithromycin is 0.03 μg/ml. For HP917, the MIC of metronidazole is 4 μg/ml. A clarithromycin MIC of >1 μg/ml and a metronidazole MIC of >8 μg/ml indicate resistance. Each experiment was repeated at least three times with the same results. Values are the means ± standard deviations from one representative experiment.