Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling
Figure 5
PI3 kinase is required for N927 invasion and is recruited to caveolae.
(A) Activation of PI3K shown by phosphorylation of Akt. Whole cell lysates of Chang cells infected with either N927 (PorBIA, P−) or N138 (PorBIB, P+) at an MOI 50 for 30 min were subjected to SDS PAGE and Western blot using anti-phospho-Akt, anti-Akt and anti-Actin antibodies. (B) Chang cells were pretreated for 1 h with PI3K inhibitors LY294002 (LY, 10 µM) or Wortmannin (WM, 1 µM) and infected with N927 (MOI 10, 30 min). Adherence (white bars) and invasion (black bars) were quantified by gentamicin protection assay. The number of adherent and invasive bacteria of untreated control cells was set as 100%. The graph shows mean values ± SD of three independent experiments performed in duplicates. p<0.01: ** (C) Distribution of signaling molecules in membrane rafts of infected cells. Chang cells were subjected to subcellular fractionation after infection either with N927 (PorBIA, P−) or N138 (PorBIB, P+) (MOI 20) for 1 h (see Text S1). Flotillin was detected as marker for the membrane raft fraction (fraction 4–5) separated from most cellular proteins (fraction 8–12). Caveolin, SREC-I (multiple bands represent differentially glycosylated forms), Flotillin and PI3K, were detected by Western blot analysis. (D) CHO cells stably transfected with either SREC-I WT (CHO-SREC-I) or empty vector control (CHO-pEGFP-N1) were infected with N927 (PorBIA, P−) at an MOI of 50 or treated with 2 µg/ml acLDL (Invitrogen) for 5 min. Whole cell lysates were subjected to SDS-PAGE and Western blot analysis using anti-phospho-Akt and anti-Tubulin antibodies. (E) Graphical representation of SREC-I gonococci interaction in Caveolae. Upon infection with N927 SREC-I localizes in cholesterol, sphingolipid and caveolin-1 rich membrane rafts. PLCy1 and PI3K are recruited to phosphorylated Cav1 (Tyrosin 14) and initiate the signaling cascade leading to endocytic uptake of the gonococci. Adapted from [25].