4% of the inoculum. This value was set at 100% and the Trichostatin A cost adhesion of the other strains was determined as a percentage of wild type adhesion. The pld mutant was significantly impaired in adhesion, adhering at only 39.7% of the wild type (p < 0.05; Figure 3A). Complementation of the pld mutant with pld in trans restored adhesion to 106.9% of wild type (Figure 3A). It should be noted that the assay as performed measures both adhered bacteria and any that have subsequently invaded. However given that invasion follows bacterial adhesion, all cell-associated bacteria, whether internalized or on the cell surface, were at one point

adherent to the host cell. Figure 3 PLD expression differentially affects adhesion (A) and invasion (B) of A. haemolyticum into HeLa cells. (A) A. haemolyticum strains were added to cell monolayers, with or without 5 mM MβCD or 312 ng HIS-PLD, and allowed to adhere for 2 h at 37°C prior to washing selleckchem and recovery of cell-associated bacteria. (B) Following adhesion, cell monolayers were washed and incubated for an additional 2 h in the presence of 10 μg/ml gentamicin to kill external bacteria. Adhesion or invasion are shown as a percentage of wild type, which was set to 100%. Error bars indicate one standard deviation from the mean calculated GSK1838705A in vitro from the averages of at least three independent experiments conducted in triplicate.

Statistical significance was calculated using single factor ANOVA and p < 0.05 was considered significant. We hypothesized that A. haemolyticum PLD promoted bacterial adhesion to host cells via receptor clustering as a result of SM cleavage, leading to lipid raft signaling. Treatment of cells with 5 mM MβCD resulted in a 44.4% reduction in the adherence of wild type A. haemolyticum to HeLa cells, as compared to untreated controls (p < 0.05; Figure 3A), indicating that the loss of lipid raft rearrangement directly affected the ability

of A. haemolyticum to adhere to HeLa cells. A. haemolyticum lacking PLD appear to invade HeLa cells more efficiently The ability of wild type and pld mutants to invade host cells was also determined. Wild type A. haemolyticum invaded HeLa cells at an average of 0.24% of the adherent bacteria. This value was set at 100% and the invasion of the other strains MycoClean Mycoplasma Removal Kit was determined as a percentage of wild type invasion. The pld mutant was not impaired in invasion, and could invade significantly better at 207.1% of wild type A. haemolyticum (p < 0.05; Figure 3B). Complementation of the pld mutant led to significantly more impaired invasion than the wild type (only 33.0% of wild type; p < 0.05; Figure 3B), which probably results from a gene dosage effect of pld expressed from a multi-copy plasmid. We also examined the effect of exogenously-added recombinant HIS-PLD on bacterial adhesion and invasion. HIS-PLD significantly enhanced the adhesion of the pld mutant and returned it to wild type levels (p < 0.