title: Involvement of focal adhesion kinase in Escherichia coli invasion of human brain microvascular endothelial cells.
abstract: 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.
authors.affiliation: Division of Infectious Diseases, Childrens Hospital Los Angeles, Los Angeles, California 90027, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Reddy
authors.forename: C A
authors.initials: CA
authors.lastname: Wass
authors.forename: K S
authors.initials: KS
authors.lastname: Kim
authors.forename: D D
authors.initials: DD
authors.lastname: Schlaepfer
authors.forename: N V
authors.initials: NV
authors.lastname: Prasadarao
mesh_terms: D001921:Brain / Q000098:blood supply / Q000382:microbiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000382:microbiology*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000472:pathogenicity*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D014443:Tyrosine; D019833:Genistein; C113183:FAK-related nonkinase; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human