01) and HLHK9∆ureA/arcA1/arcA2 (p < 0.01) (Figure  3C), and the reduction trend became more pronounced after 3 and 5 h incubation (Figure  3D). At pH 2 and 3, the survival counts of HLHK9∆ureA started to decrease (P <0.05), whereas there were dramatic decreases in the survival counts of HLHK9∆arcA1/arcA2 (p < 0.001) and triple knockout

mutant HLHK9∆ureA/arcA1/arcA2 strains, which were almost completely killed (p < 0.001) (Figure  3C). These showed that the ADI pathway of L. hongkongensis played a more important role than the urease in resisting acidic environments. Intracellular survival in J774 macrophages and mRNA this website Expression level analyses Survival PF-6463922 of wild type L. hongkongensis HLHK9, HLHK9∆ureA, HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 in J774 macrophages were shown in Figure  4A. Survival of HLHK9∆ureA/arcA1/arcA2 and HLHK9∆arcA1/arcA2 in macrophages were markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9∆ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. The decrease of survival was more prominent in HLHK9∆ureA/arcA1/arcA2, compared to HLHK9∆arcA1/arcA2 (p < 0.05) and HLHK9∆ureA (p < 0.01); and in HLHK9∆arcA1/arcA2,

compared to HLHK9∆ureA (p < 0.05). Given the above results, we further investigated the expression level of ADI genes (arcA1 and arcA2) and ureA gene of wild type L. hongkongensis HLHK9 survived in macrophages using real-time quantitative RT-PCR assay. At 8 h post infection, the mRNA levels of arcA1, arcA2 and ureA genes were markedly increased BAY 11-7082 research buy compared to those at 2 h post infection (p < 0.05, p < 0.01 Avelestat (AZD9668) and p < 0.05 respectively) (Figure  4B). Figure 4 Intracellular survival assays in J774 macrophages. A, Recovery rates of wild type L. hongkongensis HLHK9, HLHK9∆ureA,

HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 in J774 macrophages. B, Expression level of ADI genes (arcA1 and arcA2) and ureA gene of HLHK9 in macrophages. Error bars represent means ± SEM of three independent experiments. An asterisk indicates a significant difference (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Survival of L. hongkongensis strains in BALB/c mice To further investigate the role of urease and ADI pathway in acid tolerance of L. hongkongensis, we compared the survival ability of HLHK9, mutant strains HLHK9∆ureA, HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 after transit through the stomach of mice. Using this mouse model, HLHK9∆ureA exhibited similar survival abilities as HLHK9 (Figure  5). In contrast, the viable counts of HLHK9∆arcA1/arcA2 and HLHK9∆ureA/arcA1/arcA2 were reduced by 1.2-log and 1.3-log respectively, compared to that of HLHK9 (p < 0.01) (Figure  5). This also indicated that the ADI pathway played a more significant role than urease in the survival of L.