The ability of anti-PLD antibodies to block PLD-mediated lipid raft rearrangement was investigated. In the absence of PLD, addition of pre-immune or anti-PLD serum did not significantly affect the number of punctate-staining cells compared to untreated HeLa cells (9.9%; negative control; Figure 2D). In the presence of PLD, 26.0% of HeLa cells displayed punctate staining (positive control; p < 0.05 compared to the negative control; Figure HDAC inhibitor mechanism 2D). In the presence of PLD, addition of pre-immune serum did not significantly affect the number of punctate staining cells as compared to the positive control (p = 0.25; Figure 2D).

In contrast, in the presence of PLD, the addition of anti-PLD antibodies significantly reduced the number of punctate staining

cells (p < 0.05 compared to the positive control; Figure 2D). Numbers of punctate staining cells under these conditions were not significantly different to untreated HeLa Selleck Akt inhibitor cells (p = 0.15; Figure 2D), indicating that the lipid raft rearrangement seen is specific to the action of PLD. Cholesterol sequestration by MβCD blocks lipid raft rearrangement by partially solubilizing GPI-anchored and transmembrane proteins [37] and preventing small lipid rafts from aggregating into larger, functional membrane platforms [20]. In the absence of PLD, only 9.9% of HeLa cells displayed punctate staining (untreated negative control; Figure 2D). Treatment of HeLa cells with 5 mM MβCD significantly reduced the amount of punctate staining cells to 7.4% (p < 0.05 compared with the negative control; Figure 2D), indicating that spontaneous lipid raft rearrangement was being inhibited. In the presence of PLD, 26.0% of HeLa cells displayed punctate staining (positive control; p < 0.05 compared to negative control; Figure 2D). Treatment of HeLa cells with MβCD significantly reduced the level of punctate staining to 10.5% (p < 0.05 compared with the positive control; Figure 2D). This value is similar to the amount of lipid raft rearrangement seen in negative control HeLa cells (9.9%; p = 0.54; Figure 2D). These

data indicate that PLD-mediated those lipid raft rearrangement can be inhibited by cholesterol sequestration. A. haemolyticum PLD is required for efficient bacterial adhesion to the host cell The ability of PLD to enhance lipid raft rearrangement may affect the interaction between the bacterium and the host cell. To test this hypothesis, wild type and pld Salubrinal clinical trial mutant A. haemolyticum were assayed for their ability to adhere to HeLa cells. A pld mutant was constructed by allelic exchange and this mutant lost its hemolytic phenotype on TS agar containing 5% bovine blood and 10% Equi Factor. Hemolysis was restored to wild type levels upon complementation with pBJ61, carrying the pld gene (data not shown). The hemolytic phenotype was not affected by the introduction of the shuttle vector alone (data not shown). The ability of wild type or the pld mutants to adhere to HeLa cells was determined. Wild type A.