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J. Biol. Chem., Vol. 278, Issue 46, 45753-45762, November 14, 2003

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Escherichia coli K-1 Interaction with Human Brain Micro-vascular Endothelial Cells Triggers Phospholipase C-1 Activation Downstream of Phosphatidylinositol 3-Kinase^*

Sunil K. Sukumaran, George McNamara¶, and Nemani V. Prasadarao||

From the Division of Infectious Diseases and ^¶Congressman Dixon Image Core, Childrens Hospital Los Angeles and the University of Southern California Keck School of Medicine, Los Angeles, California 90027

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-. Here, further, we show that phospholipase (PLC)-1 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-, the PLC-z fragment, in HBMEC inhibits PLC-1 activation and significantly blocks E. coli invasion. PI3K activation is not affected in PLC-z/HBMEC upon infection, whereas PKC- phosphorylation is completely abolished, indicating that PLC-1 is downstream of PI3K. Concomitantly, the phosphorylation of PLC-1 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-. In addition, the recruitment of PLC-1 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-1 to the cell membrane via its interaction with pleckstrin homology domain of PLC-1. Utilizing the pleckstrin homology domains of PKC- 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 GFP_Bkt 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-1 activation in a PI3K-dependent manner to increase Ca²⁺ levels in HBMEC. This is the first report demonstrating the recruitment of activated PLC-1 to the sites of bacterial entry.

Received for publication, July 10, 2003 , and in revised form, September 2, 2003.

^* This work was supported by National Institutes of Health Grants AI40567 and HD50325 (to N. V. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Division of Infectious Diseases, MS 51, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. Tel.: 323-669-5465; Fax: 323-660-2661; E-mail: pnemani{at}chla.usc.edu.

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