Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling
Figure 6
PKD1 is required for invasion of N927.
(A) Chang cells were transfected with siRNAs against PKD1 (siPKD1) and luciferase (siLuc) as control. The cells were infected with N927 (PorBIA, P−; MOI 10) for 30 min 72 h after transfection of siRNAs. Intracellular (inv., black bars) and adherent (adh., white bars) bacteria were quantified by gentamicin protection assay and the number of adherent or invasive bacteria of control cells (siLuc) was set to 100%. Shown are the means ± SD of three independent experiments performed in duplicates. p<0.01: ** (B) Knock down of PKD1 in Chang cells was verified by Western blotting. β-tubulin was used as loading control. (C) Endogenous levels of phosphorylated PKD1 (pPKD1) were assayed after infection with N927 (MOI 100) or treatment with the known activator 12-O-Tetradecanoylphorbol 13-acetate (TPA, 0.2 µM) for 30 min. Whole cell lysates were analyzed by immunoblotting using phospho-specific PKD1 antibody (detects pSer744 and pSer748). (D) PKD1 was overexpressed by transfection of PKD-HA expression construct (Addgene; [41]) in Chang cells and phosphorylation was detected as described in (C).