Figure 1 Survival of G. mellonella following infection by H. pylori strains. Kaplan-Meier survival curves of G. mellonella larvae after 24 h-96 h from injection with 1 × 104, 1 × 105, 1 × 106 and 1 × 107 CFUs of wild type strains G27 (panel A), 60190 (panel B), M5 (panel C) are shown. Kaplan-Meier
survival curves of G. mellonella larvae after 24 h-96 h from injection with 1 × 106 CFUs of wild-type H. pylori strains G27, 60190 and M5 (panel D) are shown. The data shown are means ± SEM from three independent experiments recorded for 96 h. Differences in survival were calculated using the log-rank test for multiple comparisons. Differences were considered statistically significant at P < 0.05. PBS, phosphate-buffered saline. Table 1 Lethal dose 50% of H. pylori strains in Galleria mellonella LD 50 (means ± SEM) * Strains 48 h 72 h G27 2.8 (±0.4) × buy JIB04 105 2.4 (±0.2) Selleck BTK inhibitor × 105 G27ΔcagA 3.1 (±0.04) × 106 G27ΔcagE 2.4 (±0.06) × 106 G27ΔcagPAI 2.0 (±0.01) × 106 60190 6.1 (±0.4) × 105 1.4 (±0.04) × 106 60190ΔvacA 8.2 (±0.04) × 106 60190ΔcagA 9.7 (±0.04) × 106 60190ΔcagE 9.5 (±0.06) × 106 60190Urease-negative 8.7 (±0.04) × 106 M5 12.8 (±0.3)
× 105 2.1 (±0.08) × 105 M5 ggt::aph 12.0 (±0.6) × 105 1.0 (±0.1) × 105 *The LD50 values were expressed in Colony Forming Units (CFUs). Effect of H. pylori virulence factors on killing of G. mellonella larvae
To identify bacterial virulence factors responsible for H. pylori-induced killing of G. mellonella larvae, we compared the effects of wild-type strains G27, 60190 and M5 with those of their respective mutants in selective virulence factors. The survival percentages of a group of 10 G. mellonella larvae during 72 h post-infection with 1 × 106 CFUs of bacterial suspension were analyzed. As shown in Figure 2A, the wild-type strain G27 showed a statistically significant higher virulence compared with G27ΔcagPAI, (i.e., the G27 isogenic mutant in which the entire cag PAI has been deleted), or G27ΔcagA, or G27ΔcagE (i.e., the G27 isogenic mutants in the effector DMXAA purchase protein CagA or in the regulatory protein CagE of the type IV secretion system, respectively). Indeed, we found 15% of larvae and no larvae alive after respectively 24 h PJ34 HCl and 48 h infection with wild type G27 strain, while 55%-70% and 40-45% of larvae alive after 24 h and 48 h infection with mutant strains. Moreover, the wild-type strain 60190 showed a statistically significant increased virulence compared with its isogenic mutants defective in either CagA, or CagE, or VacA as well as with its spontaneous mutant defective in urease at 48 h (Figure 2B). In contrast, there was no significant difference between wild type strain M5 and its GGT-defective isogenic mutant M5 ggt::aph at any time post-infection (Figure 2C).