Escherichia coli K-1 interaction with human brain micro-vascular endothelial cells triggers phospholipase C-gamma1 activation downstream of phosphatidylinositol 3-kinase

Escherichia coli, the most common Gram-negative bacterium that causes meningitis in neonates, invades human brain microvascular endothelial cells (HBMEC) by rearranging host cell actin via the activation of phosphatidylinositol 3-kinase (PI3K) and PKC-alpha. Here, further, we show that phospholipase (PLC)-gamma1 is phosphorylated on tyrosine 783 and condenses at the HBMEC membrane beneath the E. coli entry site. Overexpression of a dominant negative (DN) form of PLC-gamma, the PLC-z fragment, in HBMEC inhibits PLC-gamma1 activation and significantly blocks E. coli invasion. PI3K activation is not affected in PLC-z/HBMEC upon infection, whereas PKC-alpha phosphorylation is completely abolished, indicating that PLC-gamma1 is downstream of PI3K. Concomitantly, the phosphorylation of PLC-gamma1 is blocked in HBMEC overexpressing a dominant negative form of the p85 subunit of PI3K but not in HBMEC overexpressing a dominant negative form of PKC-alpha. In addition, the recruitment of PLC-gamma1 to the cell membrane in both PLC-z/HBMEC and DN-p85/HBMEC is inhibited. Activation of PI3K is associated with the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 1,4,5-trisphosphate (PIP3), which in turn recruits PLC-gamma1 to the cell membrane via its interaction with pleckstrin homology domain of PLC-gamma1. Utilizing the pleckstrin homology domains of PKC-delta and Btk proteins fused to green fluorescent protein (GFP), which specifically interact with PIP2 and PIP3, respectively, we show herein that E. coli invasion induces the breakdown of PIP2 at the plasma membrane near the site of E. coli interaction. PIP3, on the other hand, recruits the GFPBkt to the cell membrane beneath the sites of E. coli attachment. Our studies further show that E. coli invasion induces the release of Ca2+ from intracellular pools as well as the influx of Ca2+ from the extracellular medium. This elevation in Ca2+ levels is completely blocked both in PLC-z/HBMEC and DN-p85/HBMEC, but not in DN-PKC/HBMEC. Taken together, these results suggest that E. coli infection of HBMEC induces PLC-gamma1 activation in a PI3K-dependent manner to increase Ca2+ levels in HBMEC. This is the first report demonstrating the recruitment of activated PLC-gamma1 to the sites of bacterial entry.