Role of Rac1 in Escherichia coli K1 invasion of human brain microvascular endothelial cells

Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) requires the reorganization of host cytoskeleton at the sites of bacterial entry. Both actin and myosin constitute the cytoskeletal architecture. We have previously shown that myosin light chain (MLC) phosphorylation by MLC kinase is regulated during E. coli invasion by an upstream kinase, p21 -activated kinase 1 (PAK1), which is an effector protein of Rac and Cdc42 GTPases, but not of RhoA. Here, we report that the binding of only Rac1 to PAKI decreases in HBMEC upon infection with E. coli K I, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Racl in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAKl phosphorylation was observed in HBMEC expressing cAc-Racl with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-RacI/HBMEC is due to lack of phospho-MLC recruitment to the sites of E. coli entry. Taken together the data suggest that E. coli modulates the binding of Rac 1, but not Cdc42, to PAKl during the invasion of HBMEC. (c) 2005 Elsevier SAS. All rights reserved.