Involvement Of Src Family Tyrosine Kinase In Escherichia Coli K1 Invasion Of Human Brain Microvascular Endothelial Cells

ObjectEscherichia coli Kl is the most common gram - negative pathogen of neonatal meningitis. Invasion of human brain microvascular en-dothelial cell ( HBMEC) is essential to the crossing over blood - brain barriers by E. coli K1.The exploration of the mechanisms of E. coli Kl invasion will contribute to the prevention and treatment of neonatal meningitis. It will also shed light on the research of evolution, since it deals with the interaction between prokaryotic bacterial and its eukaryotic hosts.Our previous research showed that PI3K@p - Akt@LIMK@Co-filin pathway can result in reorganozation of actin cytoskeleton. Cy-toskeletal rearrangements eventually promote E, coli entry of HBMEC. Meanwhile, PI3K@Rock@LIMK@ p - Cofilin pathway can inhibit E. coli invasion of HBMEC. However, the upstream signalings of PI3K remain to be explored. Hence, we investigated the role of Src family tyrosine kinase in the signal transduction pathways.Materials1. HBMEC (Human Brain Microvascular Endothelial Cell)2. E. coli E44 strain3. Reagents for E. coli E44 Invasive assays4. Reagents for Fluorescent staining of actin5. Reagents for Western BlottingMethods1. Human Brain Microvascular Endothelial Cell Culture and pre-treatment with PP1/PP22. E. coli invasion assays3. Fluorescent staining of actin4. Western blottingResults1. Results of E. coli invasive assays showed that Src family ki-nase selective inhibitors PP1 and PP2 could abolish E. coli E44 invasion of HBMEC2. Results of actin Fluorescent staining also showed that PP1 could block E. coli E44 invasion of HBMEC3. Results of Western Blot showed that PP1 could inhibit E. coli E44 Activation of PI3K/Akt in HBMECDiscussionSrc family tyrosine kinase selective inhibitors PP1/ PP2 inhibited E. coli Kl invasion of HBMEC in a dose - dependent manner. The results of fluorescent staining showed that in HBMEC pre - treated by PP1/ PP2, the percent of actin reorganization decrease. The results of Western Blot showed that PI3K/Akt was activated in HBMEC stimulated by invasive Escherichia coli K1; PP1/ PP2 abolished E. coli Kl activation of PI3K/Akt in HBMEC. Theses results indicated that Src family kinase is involved in E. coli Kl invasion of HBMEC; Src familyactivates PI3K/Akt in the events of cytoskeleton rearrangement and signaling molecule - structural proteins - cytoskeleton linkage.ConclusionSrc family protein tyrosine kinase is involved in E. coli Kl invasion of Human Brain Microvascular Endothelial CellsSrc family kinase activates PI3K/Akt in the events of E. coli Kl invasion of Human Brain Microvascular Endothelial CellsWe proposed a model in which Src kinase, PI3K and downstream effectors play roles in the signaling of E. coli invasion of HBMEC...