Involvement Of IbeA In Meningitic Escherichia Coli K1-Induced Polymorphonuclear Neutrophil Transmigration Across Brain Endothelial Cells

Transmigration of neutrophil (PMN) across the blood-brain barrier (BBB) is a criticalevent in the pathogenesis of bacterial meningitis. However, as a "double-edge sword", therecruitment of PMN into the CNS by transmigration across the BBB is not only crucial forhost defense against meningitic bacterial pathogens, but it is also responsible for significantCNS tissue damage, which results in devastating neurologic sequelae. Although thedevelopment of vaccines could reduce the rate of infection, but this cannot eliminatemeningitis completely. Research on bacterial pathogenecity has identified an importantvirulence factor, IbeA, that contributes to E. coli K1invasion of both intestinal epithelial cellsand BMEC both in vitro and in vivo, and BMEC is the major component of theblood-brain-barrier. The ibeA gene is a unique sequence on the GimA genetic island, onlypresent in pathogenic E. coli K1strains. However, little is know about the role of IbeA inPMN transmigration across the BBB. In this study, we investigate whether and how IbeA isinvolved in the process of PMN transmigration across the BBB.A number of in vitro and in vivo experiment models were constructed. The ability ofthe ibeA-deletion mutant strain ZD1, the ibeA gene restored-mutant, purified IbeA protein,IbeA protein coated beads, and the parent strain E44to induce PMN transmigration acrossthe brain endothelial to CNS was investigated. It was found that the ibeA-deletion mutantstrain ZD1was significantly less active in stimulating PMN transmigration than the parentstrain E44, ZD1was fully complemented by the ibeA gene or its product both in vitro and invivo. The results indicate that IbeA contributes to PMN transmigration across the BBB.After showing the involvement of IbeA in the recruitment of neutrophil to CNS duringmeningitis, the signal transduction and its molecular mechanism was investigated further.The interaction between ibeA+E. coli strains and brain endothelial cells was not resultedfrom the toxin secretion during the autolysis of bacteria. The transmigration of PMN acrossBMECs stimulated by ibeA+E. coli strains was blocked by WFA, an inhibitor on vimentin,in a dose-dependent manner. The overexpression of vimentin on human BMECs led to agreat increase of E. coli-induced neutrophil across of brain endothelial cells, and thisphenomenon did not occur to the ibeA-deletion mutant strain. The transmigrating neutrophilwas found in the net of vimentin under the confocal immunofluoresecence microscopy. Allthese indicate that vimentin contributes to the traversal of PMN across brain endothelialsstimulated by IbeA.In order to migrate to the site of infection, the leukocytes need to adhere to the blood vascular, transendothelially migrate and penetrate into the inner tissue. Previous studies ontransmigration of PMN across the BBB have been performed only to bacterial meningitiscaused by Meningococcus, Pneumococcus and Group B Streptococcus. On the other hand, E.coli-induced adhesive interactions between transmigrating leukocytes and pulmonaryendothelial cells are well understood. ICAM-1and CD44play a role in the leukocytetransmigration process during E. coli pneumonia. However, there are no in vitro and in vivostudies to date that focus on the PMN-brain endothelial cell interactions in response tomeningitic E. coli K1and its virulence factors. Surface adhesion molecules ICAM-1andCD44were upregulated after exposure of the endothelial monolayer to intact E. coli, orpurified IbeA (decontaminated with LPS). The adhesion of PMN to human BMEC wasaffected by IbeA protein in dose and time-course dependent manner. The upregulation ofICAM-1and CD44was increased by overexpression of vimentin on human BMEC anddecreased by deletion of the vimentin head domain. Three lipid rafts detergents were used inthe study of PMN migration, and all of them reduced the ration of PMN transmigration acrossBMEC in a dose-dependent manner. This suggested that there is a relationship between thecomplicity of lipid rafts/caveolae and neutrophil transmigration across BMEC in response tomeningitic E. coli.Finally, the molecular structure of IbeA protein was analyzed with bioinformatics tools,and no conserved domain was found in the secondary structure. On the basis of the predictedsecondary structure, ibeA was subcloned into four different fragments. Each fragment wasexpressed and proteins purified. It was found, by in vitro PMN transmigration assays, that F1,F2and F4-IbeA proteins did not have the ability to induce PMN transmigration across BMEC,but F3-IbeA did. This result suggested that F3-IbeA is the functional fragment of the IbeAprotein, and the sequence of amino acid from281to370is critical for IbeA to induced PMNtransmigration across BMEC.