haemolyticum strains were compared to this. Staurosporine (1 μM), used as a positive control, was able to induce apoptosis, as measured by 2.76-fold, 1.27-fold and 1.56-fold increases in caspase 3/7, 8 and 9 activities, NVP-LDE225 manufacturer respectively (p < 0.05; Figure 5). HeLa cells inoculated with wild type A. haemolyticum displayed no increase in apoptosis, as measured by caspase 3/7 or 9 activity (1.12-fold and 0.95-fold increases, respectively; Figure 5). However, HeLa cells inoculated with wild type A. haemolyticum had significantly reduced caspase

8 activity when compared to untreated cells (0.54-fold activity; p < 0.05; Figure 5). HeLa cells inoculated with the pld mutant also displayed similar levels of caspase 3/7, 8 and 9 expression as the

uninoculated HeLa cells (0.85-fold, 1.Proteasome purification 06-fold and 0.77-fold, respectively; Figure 5). The caspase 3/7 assay was repeated at 1 or 24 h post-invasion, however, no significant differences were observed in activity of these caspases at these time points (data not shown). Therefore, JNK-IN-8 purchase it appears that invasion of HeLa cells with A. haemolyticum strains was unable to induce apoptosis under these conditions (Figure 5). Figure 5 Intracellular PLD does not initiate apoptosis in HeLa cells. HeLa cells were inoculated with A. haemolyticum strains and the bacteria were allowed to adhere for 2 h and invade for 5 h prior to measurement of caspase 3/7, 8 or 9 activity. Activity Demeclocycline is shown as a fold-change of untreated cells, which was set at a nominal value of 1.0. Error bars indicate one standard deviation from the mean calculated from the averages of at least three independent experiments conducted in triplicate. As bacterial invasion did not induce apoptosis, it suggested that loss of HeLa cell viability may be due to necrosis. HeLa cells were inoculated with A. haemolyticum strains and examined by TEM. Uninoculated, control HeLa cells displayed normal architecture (Figure 6A). HeLa cells inoculated with the pld mutant displayed typical cellular architecture; however, bacteria could

be observed in membrane-bound vacuoles within some cells (Figure 6B). In contrast, wild type inoculated cells appeared necrotic, as there was no membrane integrity, the cytoplasm appeared to be absent, the nucleus was condensed and the mitochondria were swollen (Figure 6C, D), all of which are hallmarks of cellular necrosis. Bacteria could be observed both in proximity to, and inside, the HeLa cells, and intracellular bacteria were not found within vacuoles (Figure 6C). Figure 6 PLD apparently induces host cell damage by necrosis. Representative transmission electron micrographs of HeLa cells, (A) uninoculated, or inoculated with (B) A. haemolyticum pld mutant or (C, D) A. haemolyticum wild type using a standard invasion assay. Arrows indicate bacteria, N and M indicate the nucleus and mitochondria, respectively.