Tet-regulated silencing of urease expression during active infection.

Urease expression in conditional urease mutant strains OND3241 was induced with 50 ng/ml ATc for 48 h prior to oral challenge. (A) Mice were orally challenged with pre-induced OND3241 and supplemented with 5 mg/l ATc for two weeks. ATc supplement was maintained (+, ■) or withdrawn (+/-, □) and animals were sacrificed 0, 1, 3, 5 and 7 days later. Bars represent median bacterial load per group (n = 5) and points plotted represent colonization density for each individual animal. Detection limit was < 50 CFU per stomach (dotted horizontal line). Gastric specimens without H. pylori re-isolation are shown as null. (B) Mice challenged with wild-type X47 (WT, ●) or pre-induced OND3241 were supplemented with 5 mg/l ATc for two weeks. After this two week period, ATc supplement was maintained (+, ■) or withdrawn (+/-, □) and animals were sacrificed 3, 5 and 42 days later. Bars represent median bacterial load per group (n = 10) and points plotted represent colonization density for each individual animal. Detection limit was < 50 CFU per stomach (dotted horizontal line). Gastric specimens without H. pylori re-isolation are shown as null. Statistical analysis using the unpaired two-sided Mann-Whitney test are indicated above the compared groups (* indicates that the geometric value of the (+/-, □) group is significantly different (p ≤ 0.05) from the (+, ■) group of the same time point) and unpaired two-sided Fisher’s exact test analysis of the infection rate by the conditional mutant is presented below the compared groups (* p< 0.05; *** p < 0.001). For the day 42 time point in panel B, as all the analysed colonies isolated from the (+/-, □) group were found to be escape mutants, none of the animals were considered infected with the conditional mutant resulting in a 0/10 infection rate.