Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5 rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Escherichia coli K1 is the most frequent causative agent of neonatal meningitis. The pathogenic mechanisms of E. coli have been studied by utilizing brain microvascular endothelial cells (BMEC) as an in vitro blood-brain barrier (BBB) model (17,19,20). These studies suggest that S fimbriae mediate attachment to BMEC via NeuAc2,3-Gal epitopes of BMEC surface glycoproteins; however, they do not play a significant role in invasion (25). More-intimate attachment by the bacterial outer membrane protein A (OmpA) mediates the invasion process (15). A similar phenomenon has been identified in the pathogenesis of Neisseria gonorrhoeae, where pili promote initial adherence followed by Opa-mediated interaction for invasion (10). In addition to OmpA, other bacterial factors such as IbeA, IbeB, TraJ, and CNF also play roles in E. coli invasion of BMEC; however, OmpA appears to be the most important factor (2, 6, 7, 9). OmpA ϩ E. coli induces actin rearrangements at the site of bacterial entry, which are completely abolished by treatment of the bacteria with GlcNAc␤1-4GlcNAc polymers, which are receptor analogs (16,17). Computer simulation studies of the interactions between OmpA and GlcNAc␤1-4GlcNAc epitopes indicate that these sugars have more favorable energy than any other sugar molecule tested in our experiments (4). These results are in good agreement with earlier studies in which GlcNAc␤1-4GlcNAc moieties showed significant blocking of E. coli invasion both in vitro and in vivo (16).In support of the role of OmpA in E. coli K1 invasion, studies have also demonstrated that OmpA ϩ E. coli induces the phosphorylation of focal adhesion kinase (FAK) and its interaction with phosphatidylinositol 3-kinase (PI3K) (20, 21). Furthermore, GlcNAc␤1-4GlcNAc polymers blocked the activation of FAK, although at higher concentrations, indicating the role of the human BMEC (HBMEC) receptor for OmpA in transducing signals for internalization of E. coli. In addition, it was shown that E. coli also induces the activation of protein kinase C alpha (PKC-␣) in an OmpA-dependent manner (27). The activated PKC-␣ is recruited to the plasma membrane, where it interacts with caveolin-1, a protein marker for caveolae, for internalization of E. coli (28). Several receptors, such as epidermal growth factor and fibroblast growth factor (14), have been shown to accumulate in caveolae, suggesting that the OmpA receptor could be part of caveolae during...

Cytoskeletal dynamics, modulated by actin-myosin interactions, play an important role in Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC). Herein, we show that inhibitors of myosin function, butanedione monoxide and ML-7, significantly blocked the E. coli invasion of HBMEC. The invasive E. coli induces myosin light-chain (MLC) phosphorylation during the invasion process, which gets recruited to the site of actin condensation beneath the bacteria. We also show that invading E. coli downregulates the activity of p21-activated kinase 1 (PAK1), which is an upstream regulator of MLC kinase (MLCK). Overexpression of wild-type PAK1 and constitutively active PAK1 in HBMEC inhibits E. coli invasion significantly with a concomitant decrease in MLC phosphorylation. The inhibition of E. coli invasion by these PAK1 mutants is due to the absence of phospho-MLC at the actin condensation points. In contrast, the dominant-negative PAK1 shows no effect either on the invasion or on MLC phosphorylation or phospho-MLC recruitment to the actin focal points, suggesting that activated PAK1 inactivates MLCK. Taken together, these results suggest that E. coli invasion of HBMEC induces MLC phosphorylation by inhibiting the activity of PAK1 and the recruitment of phosphorylated MLC to the site of actin condensation beneath the bacteria for efficient internalization of E. coli into HBMEC.

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 PAK1 decreases in HBMEC upon infection with E. coli K1, which resulted in increased phosphorylation of MLC. Overexpression of a constitutively active (cAc) form of Rac1 in HBMEC blocked the E. coli invasion significantly, whereas overexpression of a dominant negative form had no effect. Increased PAK1 phosphorylation was observed in HBMEC expressing cAc-Rac1 with a concomitant reduction in the phosphorylation of MLC. Immunocytochemistry studies demonstrated that the inhibition of E. coli invasion into cAc-Rac1/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 Rac1, but not Cdc42, to PAK1 during the invasion of HBMEC.