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Infection and Immunity, November 2000, p. 6423-6430, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Involvement of Focal Adhesion Kinase in Escherichia coli Invasion of Human Brain Microvascular Endothelial Cells

Marpadga A. Reddy,1 Carol A. Wass,1 Kwang Sik Kim,1,2 David D. Schlaepfer,3 and Nemani V. Prasadarao1,2,*

Division of Infectious Diseases, Childrens Hospital Los Angeles,1 and University of Southern California School of Medicine,2 Los Angeles, California 90027 and Department of Immunology, The Scripps Research Institute, La Jolla, California 920373

Received 16 May 2000/Returned for modification 14 July 2000/Accepted 11 August 2000

Escherichia coli K1 traversal across the blood-brain barrier is an essential step in the pathogenesis of neonatal meningitis. We have previously shown that invasive E. coli promotes the actin rearrangement of brain microvascular endothelial cells (BMEC), which constitute a lining of the blood-brain barrier, for invasion. However, signal transduction mechanisms involved in E. coli invasion are not defined. In this report we show that tyrosine kinases play a major role in E. coli invasion of human BMEC (HBMEC). E. coli induced tyrosine phosphorylation of HBMEC cytoskeletal proteins, focal adhesion kinase (FAK), and paxillin, with a concomitant increase in the association of paxillin with FAK. Overexpression of a dominant interfering form of the FAK C-terminal domain, FRNK (FAK-related nonkinase), significantly inhibited E. coli invasion of HBMEC. Furthermore, we found that FAK kinase activity and the autophosphorylation site (Tyr397) are important in E. coli invasion of HBMEC, whereas the Grb2 binding site (Tyr925) is not required. Immunocytochemical studies demonstrated that FAK is recruited to focal plaques at the site of bacterial entry. Consistent with the invasion results, overexpression of FRNK, a kinase-negative mutant (Arg454 FAK), and a Src binding mutant (Phe397 FAK) inhibited the accumulation of FAK at the bacterial entry site. The overexpression of FAK mutants in HBMEC also blocked the E. coli-induced tyrosine phosphorylation of FAK and its association with paxillin. These observations provide evidence that FAK tyrosine phosphorylation and its recruitment to the cytoskeleton play a key role in E. coli invasion of HBMEC.

* Corresponding author. Mailing address: Division of Infectious Diseases, Mail Stop 51, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. Phone: (323) 669-5465. Fax: (323) 660-2661. E-mail: pnemani{at}chla.usc.edu.

Infection and Immunity, November 2000, p. 6423-6430, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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