This plasmid was used to transform A. haemolyticum ATCC9345, selecting for KnRCmS colonies. Southern blot analysis of A. haemolyticum wild type and pld- mutant genomic DNA confirmed inactivation of the pld gene via a

double cross-over event (data not shown). A pld complementing plasmid, pBJ61, CAL-101 was constructed by cloning the insert of pBJ29 into pJGS180 [43], which replicates in A. haemolyticum (data not shown). Tissue culture cell adhesion and invasion assays HeLa cells were cultured in Iscove’s Modified Dulbecco’s Medium with 10% fetal calf serum (IMDM-10% FCS) with 10 μg/ml gentamicin at 37°C and at 5% CO2. For adhesion assays, cells in IMDM-10% FCS, without gentamicin, were seeded into 24-well Crenigacestat plates at 2 × 105 cells/well in 1 ml volumes. The cells were incubated overnight prior to the addition of log-phase A. haemolyticum at a multiplicity of infection (MOI) of 10:1. Bacterial adhesion was assessed after 2 h at 37°C. Cell monolayers were washed three times with 0.1M phosphate-buffered saline, pH 7.2 (PBS) to remove non-adherent bacteria. Cell monolayers were lysed using 1 ml ice-cold 0.1% Triton X-100 for 10 min, and viable bacteria were enumerated by dilution plating. To assess the inhibitory affect of the cholesterol sequestering agent methyl-beta-cyclodextrin (MβCD; Sigma) on adhesion, 5 Ralimetinib mM MβCD

was added to HeLa cells for 40 min prior to addition of bacteria, as described above, and maintained at 5 mM in the medium for the duration of the experiment. To assess the effect of exogenous PLD, 312 ng HIS-PLD was added to HeLa cells for 10 min prior

to the addition of bacteria, as described above. For invasion assays, bacteria were added at an MOI of 20:1, were allowed to adhere and invade for 2 h, at which time the cell monolayers were washed three times with Hank’s Balanced Salt Solution, and IMDM-10% FCS containing 10 μg/ml gentamicin was added to the wells. The plates were incubated for an additional 2 h to allow invasion and killing of extracellular bacteria. The monolayers were washed and internalized bacteria were recovered and enumerated as above. Epithelial cell cytotoxicity The cytotoxicity of HIS-PLD for epithelial cells was determined using the CellTiter 96® Aqueous One Solution Etomidate Cell Proliferation Assay (Promega). HeLa cells were seeded into 96-well plates at 2 × 104 cells/well and the cells were incubated for 18 h to achieve 80% confluence. Triplicate wells were incubated with doubling dilutions of HIS-PLD (0-2 μg) and incubated for 2-24 h, as above. Dilutions of imidazole-containing HIS-protein elution buffer were used as a control. Additional monolayers were inoculated with log-phase A. haemolyticum strains at an MOI of 20:1, and incubated for 2 h, as above. The monolayers were washed three times with PBS and IMDM-10% FCS containing 10 μg/ml gentamicin was added and the cells were incubated for a further 5 h.