37-kDa Laminin Receptor Precursor Modulates Cytotoxic Necrotizing Factor 1–mediated RhoA Activation and Bacterial Uptake

Jin Woong Chung,Suk Jin Hong,Kee Jun Kim,Daniel Goti,Monique F Stins,Sooan Shin,Valina L Dawson,Ted M Dawson,Kwang Sik Kim

doi:10.1074/jbc.m301028200

Abstract

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells.

Highlights

Escherichia coli K1 is a major cause of neonatal Gramnegative bacillary meningitis
We have previously shown that E. coli invasion of human brain microvascular endothelial cells (HBMEC)1 is a prerequisite for penetration into the central nervous system in vivo, and identified several E. coli determinants contributing to invasion of HBMEC, including Ibe proteins, AslA, TraJ, and Cytotoxic necrotizing factor 1 (CNF1) [1,2,3,4,5,6]
A yeast colony co-transformed with pGADT7:laminin receptor precursor (LRP)(89 –295) and pGBKT7:N-terminal domain of CNF1 (nCNF1) was positive in both growth on the selection medium (Leu, Trp, His, Ade-) and 5-bromo-4chloro-3-indolyl-␤-D-galactopyranoside (X-gal) assays (Fig. 1a)

Summary

Introduction

Escherichia coli K1 is a major cause of neonatal Gramnegative bacillary meningitis. Despite advances in antimicrobial chemotherapy and supportive care, the mortality and morbidity associated with E. coli meningitis remain significant because of incomplete understanding of the pathogenesis of this disease. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRPoverexpressing cells.

Results

Conclusion