Immediately after elimination of extracellular bacteria by gentamicin treatment (0 h post gentamicin treatment), no statistically significant difference was observed in the counts of internalized wild-type or htrA mutant bacteria (Figure 3A), with 0.24 and 0.18% of the original inoculum recovered, respectively.
The counts of internalized bacteria recovered 5 h post gentamicin treatment decreased significantly to 0.08 and 0.025% of the original inoculum for the wild-type and the htrA mutant, respectively. This decrease in intracellular survival was significantly greater for the htrA mutant (~7 fold) selleck chemicals compared to the wild-type strain (~3 fold) (Figure 3A). While no htrA mutants were detected at 24 h, ~1 × 103 CFU/ml of wild-type bacteria were recovered at this time point, YH25448 supplier representing a ~300 fold reduction
compared with the 0 h time point. These data indicate that htrA is important for intra-amoebae survival in the 24 h time frame studied, but not for the uptake step. This suggests that pre-exposure to stress, via its transcriptional regulation on virulence-associated genes, may affect survival of intra-amoeba bacteria. Figure 3 Intracellular survival rates of C. jejuni cells within A. castellanii . Intracellular survival rates were determined by colony PX-478 forming unit (CFU) counting at 0, 5, and 24 h post gentamicin treatment at 25°C in aerobic conditions. Panel A: comparison of wild-type (WT) and htrA mutant. Panel B: comparison of stressed and non-stressed wild-type bacteria. until Data are means and standard errors of three independent experiments. Statistically significant differences concern comparisons between control and treatment groups. (*) p < 0.05; (**) p < 0.01; nd, none detected. Uptake of stressed C. jejuni by A. castellanii and intracellular survival To examine the impact of pre-exposure to stressful environments on the degree of phagocytosis by amoebae
and on the intracellular survival of wild-type C. jejuni in amoebae, stressed and non-stressed C. jejuni cells were co-cultured with A. castellanii. Approximately 4.5 × 108 CFU/ml bacteria were subjected to either the stress or control treatments before interactions with amoeba. The survival data presented in Figure 3B were normalized to account for the number of bacteria that had survived exposure to the stress tested (or to the control treatment) before inoculation of the amoeba. Immediately after elimination of extra-amoeba bacterial cells by gentamicin treatment, approximately 0.18% of the original non-stressed bacterial inoculum was recovered as internalized bacteria, but only ~0.06 and 0.14% of the C. jejuni inoculum pre-exposed to low nutrient and osmotic stresses were recovered, respectively (Figure 3B). No statistically significant differences were obtained with C. jejuni pre-exposed to heat and oxidative stresses compared with non-stressed bacteria.