06% of the original inoculum was obtained for non-stressed C. jejuni. Pre-exposure of bacteria to heat, starvation or osmotic stresses exacerbated the bacterial susceptibility to intracellular killing, since a significant
decline of the number of surviving bacteria was observed upon pre-exposure to these stresses 5 h post-gentamicin treatment (Figure 3B). At 24 h post gentamicin Palbociclib cell line treatment, a few internalized bacteria (~1.5 × 103 CFU/ml) were observed with non-stressed inoculum. No bacteria that had been pre-exposed to heat, starvation or osmotic stress were detected. In contrast, pre-exposure to oxidative stress had no impact on internalization or intracellular survival of C. jejuni under the conditions and time frame studied. Effect of pre-exposure to stress on sub-cellular AZD6244 molecular weight location of internalized bacteria A detailed observation of C. jejuni cells internalized within the amoebae was carried out by confocal laser scanning microscopy (CLSM). In the absence of any stress, live C. jejuni cells were detected by CellTracker Red staining inside the trophozoites immediately after gentamicin treatment (Figure 4A, B). The intracellular bacteria were distributed as clusters within acidic vacuoles as
observed by the simultaneous staining of acidic vacuoles by LysoSensor Green DND-189 (Figure 4C, D). Pre-exposure of bacteria to low-nutrient, heat, osmotic or oxidative stresses did not qualitatively alter the sub-cellular location of internalized bacteria, as all were also recovered in acidic vacuoles (Figure 4E to T). Figure 4 Confocal microscopy
analysis of stressed and non-stressed C. jejuni cells within acidic organelles of A. castellanii observed immediately after gentamicin treatment. Control Thiamet G C. jejuni (A-D), C. jejuni pre-exposed to osmotic stress (E-H), heat stress (I-L), hydrogen peroxide (M-P), or starvation stress (Q-T). The multiplicity of infection was 100:1 (bacteria:amoeba). (A, E, I, M, Q) differential interference contrast image; (B, F, J, N, R) C. jejuni stained with CellTracker Red; (C, G, K, O, S) acidic amoeba organelles stained with LysoSensor Green; (D, H, L, P, T) corresponding overlay. Scale bar = 5 μm. In addition to the viable count assay for the quantification of intracellular bacteria and CLSM analyses reported above, TEM was also used to more precisely assess the effect of heat stress on intracellular location of C. jejuni within A. castellanii. Heat stress was selected for TEM studies because it decreased intracellular survival of C. jejuni, but it did not affect uptake. Therefore this heat stress allowed visualization of numerous internalized bacteria at early time points. As shown in Figure 5, sections of infected A. castellanii cells obtained right after gentamicin treatment showed that C. jejuni cells were confined to tight vacuoles within the amoebae, whether they had been heat-stressed or not prior to co-culture with amoebae (Figure 5A, C).