FimH-mediated Escherichia coli K1 invasion of human brain microvascular endothelial cells Naveed Ahmed Khan, 1,2 Yuri Kim, 1 Sooan Shin 1 and Kwang Sik Kim 1 * 1 Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Park 256, Baltimore, MD 21287, USA. 2 School of Biological and Chemical Sciences, Birkbeck College, University of London, London WC1E 7HX, UK. Summary Adhesion to brain microvascular endothelial cells, which constitute the blood–brain barrier is consid- ered important in Escherichia coli K1 bacterial pen- etration into the central nervous system. Type 1 fimbriae are known to mediate bacterial interactions with human brain microvascular endothelial cells (HBMEC). Here, we demonstrate that type 1 fimbriae, specifically FimH adhesin is not only an adhesive organelle that provides bacteria with a foothold on brain endothelial cells but also triggers signalling events that promote E. coli K1 invasion in HBMEC. This is shown by our demonstrations that exogenous FimH increases cytosolic-free-calcium levels as well as activates RhoA. Using purified recombinant mannose-recognition domain of FimH, we identified a glycosylphosphatidylinositol-anchored receptor, CD48, as a putative HBMEC receptor for FimH. Fur- thermore, E. coli K1 binding to and invasion of HBMEC were blocked by CD48 antibody. Taken together, these findings indicate that FimH induces host cell signalling cascades that are involved in E. coli K1 invasion of HBMEC and CD48 is a putative HBMEC receptor for FimH. Introduction Despite advances in antimicrobial chemotherapy, the morbidity and mortality associated with bacterial meningi- tis have remained significant (Gladstone et al., 1990; Unhanand et al., 1993; Scott et al. 1998). This is due to our incomplete understanding of the pathogenesis and pathophysiology of this serious disease. Escherichia coli K1 is the most common gram-negative bacterium that causes meningitis during the neonatal period (Gladstone et al., 1990; Unhanand et al., 1993; Scott et al. 1998). Most cases of E. coli meningitis develop as a result of haematogenous spread but it is not clear how circulating bacteria cross the blood–brain barrier to gain entry into the central nervous system (CNS) (reviewed in Kim, 2006). We have successfully isolated and cultivated human brain microvascular endothelial cells (HBMEC), which constitute the blood–brain barrier (Stins et al., 1997). Our studies have demonstrated that E. coli K1 invasion of HBMEC is a prerequisite for E. coli penetration into the central nervous system in vivo. In support, we have identified several E. coli determinants (e.g. Ibe pro- teins and CNF1), which contribute to HBMEC invasion in vitro as well as penetration into the CNS in vivo (Kim, 2001, 2003). Adherence is an important first step in E. coli traversal of the blood–brain barrier. E. coli may use adhesion to HBMEC both as an initial step before proceeding to the invasion as well as to withstand blood flow in vivo. Our recent studies have shown that type 1 fimbrial adhesin, FimH, mediates E. coli K1 binding to and subsequent invasion of HBMEC (Teng et al., 2005). These results are consistent with other findings, which showed that uro- pathogenic E. coli (UPEC) strains utilize FimH to mediate bacterial attachment to the bladder epithelial cells and these interactions are crucial for the ability of UPEC strains to produce disease (Connell et al., 1996; Langer- mann et al., 1997; Thankavel et al., 1997; Mulvey et al., 1998). In this study, we determined the mechanisms asso- ciated with FimH-mediated E. coli K1 binding to and inva- sion of HBMEC. Results Purification of the mannose-recognition domain of FimH Our pilot experiments revealed that full-length FimH protein (approximate molecular weight 29 kDa) is insoluble and thus it was difficult to obtain functionally active full-length FimH (unless copurified with FimC), which was consistent with the findings of other investi- gators (P. Klemm, Technical University of Denmark, pers. comm.; Tewari et al., 1993). We therefore expressed the mannose-recognition domain of FimH (residues 1–156, approximate molecular weight 15 kDa), which was Received 8 May, 2006; revised 8 June, 2006; accepted 28 June, 2006. *For correspondence. E-mail kwangkim@jhmi.edu; Tel. (+1) 410 614 3917; Fax (+1) 410 614 1491. Cellular Microbiology (2007) 9(1), 169–178 doi:10.1111/j.1462-5822.2006.00779.x First published online 10 August 2006 © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd