67-kDa Laminin Receptor Promotes Internalization of Cytotoxic Necrotizing Factor 1-expressing Escherichia coli K1 into Human Brain Microvascular Endothelial Cells

Kee Jun Kim,Jin Woong Chung,Kwang Sik Kim

doi:10.1074/jbc.m410176200

Abstract

Escherichia coli K1 is the most common Gram-negative organism causing meningitis, and its invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for penetration into the central nervous system. We have reported previously that cytotoxic necrotizing factor 1 (CNF1) contributes to E. coli K1 invasion of HBMEC and interacts with 37-kDa laminin receptor precursor (37LRP) of HBMEC, which is a precursor of 67-kDa laminin receptor (67LR). In the present study, we examined the role of 67LR in the CNF1-expressing E. coli K1 invasion of HBMEC. Immunofluorescence microscopy and ligand overlay assays showed that 67LR is present on the HBMEC membrane and interacts with CNF1 protein as well as the CDPGYIGSR laminin peptide. 67LR was up-regulated and clustered at the sites of E. coli K1 on HBMEC in a CNF1-dependent manner. Pretreatment of CNF1+ E. coli K1 with recombinant 37-kDa laminin receptor precursor reduced the invasion rate to the level of Deltacnf1 mutant, and the invasion rate of CNF1+ E. coli K1 was enhanced in 67LR-overexpressing HBMEC, indicating 67LR is involved in the CNF1+ E. coli K1 invasion of HBMEC. Coimmunoprecipitation analysis showed that, upon incubation with CNF1+ E. coli K1 but not with Deltacnf1 mutant, focal adhesion kinase and paxillin were recruited and associated with 67LR. When immobilized onto polystyrene beads, CNF1 was sufficient to induce internalization of coupled beads into HBMEC through interaction with 67LR. Taken together, this is the first demonstration that E. coli K1 invasion of HBMEC occurs through the ligand-receptor (CNF1-67LR) interaction, and 67LR promotes CNF1-expressing E. coli K1 internalization of HBMEC.

Highlights

The successful entry of invasive bacterial pathogens into the nonphagocytic cells such as endothelial and epithelial cells is the key to the establishment of several infectious diseases, including bacterial meningitis
The cnf1 Deletion Mutant (⌬cnf1) of E. coli K1 Is Defective in Invasion but Not in Binding to human brain microvascular endothelial cells (HBMEC)—cytotoxic necrotizing factor 1 (CNF1) has been shown to contribute to E. coli K1 invasion of HBMEC in vitro and traversal of the blood-brain barrier in the experimental hematogenous meningitis animal model [9]
CNF1 Interacts with 67-kDa laminin receptor (67LR) of HBMEC—We have shown previously that the 37-kDa laminin receptor precursor (37LRP) of HBMEC interacts with CNF1 of E. coli K1, and its expression level affects the activities of CNF1 such as RhoA activation and bacterial uptake [10]. 37LRP is shown to be a cytoplasmic protein and matured to 67LR, which is known to be present on the cell surface and binds to laminin with high affinity [17]

Summary

Introduction

The successful entry of invasive bacterial pathogens into the nonphagocytic cells such as endothelial and epithelial cells is the key to the establishment of several infectious diseases, including bacterial meningitis. CNF1ϩ E. coli K1 Induces 67LR Clustering around the Bacteria on the Cell Surface of HBMEC—We have reported previously [9, 10] that CNF1 is a major bacterial determinant contributing to E. coli K1 invasion of HBMEC.

Results

Conclusion