Escherichia coli K1-induced cytopathogenicity of human brain microvascular endothelial cells Naveed Ahmed Khan * , Junaid Iqbal, Ruqaiyyah Siddiqui Department of Biological and Biomedical Sciences, Aga Khan University, Stadium Road, Karachi, Pakistan article info Article history: Received 24 May 2012 Received in revised form 2 July 2012 Accepted 3 July 2012 Available online 20 July 2012 Keywords: Sepsis Endothelial cells Lipopolysaccharide Cytopathogenicity abstract Pathophysiology of Escherichia coli sepsis is complex involving circulating bacterial products, cytokine release, and sustained bacteremia resulting in the damage of vascular endothelium. Here, it is shown that E. coli K1 produced cytopathogenicity of human brain microvascular endothelial cells (HBMEC), that constitute the bloodebrain barrier. Whole bacteria or their conditioned medium produced severe HBMEC damage suggesting E. coli K1-cytopathogenicity is a contact-independent process. Using lipo- polysaccharide (LPS) inhibitor, polymyxin B, purified LPS extracted from E. coli K1 as well as LPS mutant derived from E. coli K1, we showed that LPS is not the sole determinant of E. coli K1-mediated HBMEC death. Bacterial product(s) for HBMEC cytopathogenicity was heat-labile suggesting LPS-associated proteins. Several isogenic gene-deletion mutants (DompA, DibeA, DibeB, Dcnf1) exhibited HBMEC cyto- pathogenicity similar to that produced by wild type E. coli K1. E. coli K1-mediated HBMEC death was independent of phosphatidylinositol 3-kinase (PI3K) but dependent partially on focal adhesion kinase (FAK) using HBMEC expressing dominant negative FAK and PI3K. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Escherichia coli is a Gram-negative bacterium, a causative agent of septicemia and meningitis with fatal consequences. Although the pathophysiological events in these diseases differ remarkably, a high level of bacteremia is a pre-requisite in both E. coli-mediated sepsis and meningitis [1]. Research in sepsis has demonstrated that pathophysiological events during the course of Gram-negative septic shock are primarily elicited by lipopolysaccharide (LPS) [2], as characterized by high concentrations of endotoxin and excessive cytokines release [3e6]. In vitro data showed that purified LPS from pathogenic E. coli produced cell death in bovine endothelial cells [7,8]. Taken together, these findings signified LPS as a potential candidate to design therapeutic interventions against E. coli infec- tions. However, anti-LPS strategies explored to date have failed to prevent the clinical course of Gram-negative sepsis, [9e11] which raises the question whether LPS is the sole toxic element in Gram- negative sepsis [12]. With regards to meningitis, E. coli traversal of the bloodebrain barrier is shown to be the key step in the development of E. coli K1 meningitis [13,14]. Using human brain microvascular endothe- lial cells (HBMEC), which constitutes the bloodebrain barrier, several determinants (such as OmpA, IbeA, IbeB, CNF1) are shown to be important for E. coli K1 binding to and invasion of HBMEC, leading to bacterial crossing of the bloodebrain barrier endothelium. Here, we studied the ability of E. coli K1 to produce HBMEC cytopathogenicity and determined the requirement of several bacterial determinants that are known to play crucial roles in E. coli K1 crossing of the bloodebrain barrier endothelium. In addition, we studied the role of signaling molecules including protein tyro- sine kinases, phosphatidylinositol 3-kinase (PI3K) and focal adhe- sion kinase (FAK) in E. coli K1-mediated HBMEC cytopathogenicity. 2. Materials and methods 2.1. Human brain microvascular endothelial cell (HBMEC) cultures and transfections HBMEC were routinely grown on rat tail collagen-coated dishes in growth medium [RPMI containing 10% heat-inactivated fetal bovine serum, 10% Nu-serum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 U/ml), streptomycin (100 mg/ml), non-essential amino acids and vitamins] as previously described [15,16]. The HBMEC expressing dominant negative forms of PI3K (Dp85 or Dp110) and FAK (Y397F construct in a pcDNA3 plasmid) were kindly provided by K. S. Kim (Johns Hopkins University, USA) and cultured as previously described [17,18]. * Corresponding author. Tel.: þ92 (0) 21 3486 4540; fax: þ92 (0) 21 3493 4294. E-mail address: Naveed5438@gmail.com (N.A. Khan). Contents lists available at SciVerse ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath 0882-4010/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.micpath.2012.07.001 Microbial Pathogenesis 53 (2012) 269e275