Function Analysis Of The Genes Of Invasion Of HBMEC Of E. Coli In Neonatal Meningitis

PurposeEscherichia coli K1 is the most common blood - spread Gram - negative organism causing neonatal meningitis, but it is incompletely understood how E. coli Kl crosses the blood - brain barrier (BBB). To study the molecular mechanism of Escherichia coli. trafficking is not only helpful to understand pathogenesity of the bacteria, but also valuable for us to develop novel strategies to prevent bacteria meningitis. meanwhile, throught the study of HBMEC we can get some useful data about BBB , which may be valuable for the treatment of brain disease. At present,the leading group in this field is the group of Kim KS. Their contributions to this field are: (1) successful seperation HBMEC and establishment of in vitro model of BBB. (2)they haved found many genes contributes to Escherichia coli invasion of BBB such as yijP, ibeA, ibeB,aslA ,compA ,ypdP etc. ,they also found the attachment of Escherichia coli to HBMEC can cause the arrangement of host cell and the phosphorylation of FAK and activation of PI3K signaling pathway. Recently, they found that there may be a few independent signalling pathway during Escherichia coli invasion of BBB,It is characteristic of bacteria invasion causing the cytoskeletal rearrangement of host cell. Through this change bacteria facilitate the absorption, invasion processes of themselves. In recent years the mechanism of A/E type bacteria is almost known, for example, the enterobacteria Escherichia coli Esp proteins are the main components of type - HI secretion system , also take part in cytoskeletal reorganization of host cell. Now, we know Escherichia coli Kl can also cause the cytoskeletal rearrangement of HBMEC , but the effector protein is so far not known,The purpose of this study is :to make clear the exact functionof each ibe protein in the host cell invasion process, through the study of cy-toskeletal change , binding rate change, invasion rate change , we may determine the function of invasion gene products and the associated signaling pathway.Method1. Deletion of ibeB gene by gene knock - out method. the DNA fragment in which ibeB gene was partly deleted was cloned into selfsuicidal plasmid pCDV442 ,the recombinant plasmid pCVD7D was transferded into E44 by conjugation method, through antibiotic and sucrose seletion we got ibeB gene deletion mutant. The mutant was checked by PCR amplification of ibeB gene.2. Cytoskeletal change of HBMEC is determined by rohdamine pholloidin stain. The E44 and single gene deleted mutants E44:â–³ibeA, E44:â–³ibeB, E44: â–³ibeC were cultured and harvested ,the bacteria added to 50% confluent HBMEC cultivate at 37C⁰ for 30 minutes,then wash away the bacteria fix the HBMEC with 4% freshly made formal dehyde, stain the HBMEC with rohodamine pholloidin , check the Cytoskeletal change of HBMEC under microscope .3. The effect of invasion gene deletion on bacteria invasion rate to HBMEC. Invasion experiment: The single gene deleted mutants and E44 were grouped as follows: E44, E44: â–³ibeA, E44â–³: ibeB, E44: â–³ibeC, ( E44: â–³ibeA, E44: â–³ibeB) ( E44: â–³ibeC E44: â–³ibeA) , ( E44: â–³ibeB, E44: â–³ibeC ) and negative control . The harvested bacteria were added to the confluent HBMEC incubate at 37C⁰ for 1 hour ,wash away the suspended bacteria ,add genta-micin containing media incubate at 37C⁰ for 1 more hour, removed the media lysed HBMEC with water count the bacteria in the lysate on BHI plate, calculate the invasion rate of each group.4. Gene transfection: transfect HBMEC with p^cDNA7c select positive clone with G418. Isolate DNA and RNA of positive clone cell as tempate detect ibeB gene and its expression using PCR and RT - PCR5. Western blot assay: The ibeB transfected HBMEC were cultered and harvested . Isolate the protein of harvested cell , and detect the change of Akt and p- Akt by Western blot method6. Cell immunoflurecent assay: The ibeB transfected HBMEC were cultured to confluency, the cell were fixed with 4% formal dehyde, the treated HBMEC were incubated, with zo - 1 antibody after wash stained the HBMEC with the second fluorescent antibody.Results1. Through deletion manipulation and PCR verification we got the ibeB gene deletion mutant . named E44: â–³ibeB2. The invasion rate of each bacteria are as follows: E44:3.46 ± 0. 65 E44: â–³ibeA :0. 54 ±0. 17 E44:â–³ibeB(?)0. 82 ±0.28 E44:â–³ibeC: 0. 73 ±0. 17The analysis of coopertivity each bacteria mutant are as follows: (E44: â–³ibeA E44:â–³ibeB) 100 ± 4.6; (E44:â–³ibeB E44:AibeC) 44 ± 9; (E44:â–³ibeA E44:â–³ibeC 76 ± 223. There are different change in cytoskeleton and tight junction of HBMEC infected by wild type and single gene deleted mutants: (1) E44 infected HBMEC showed remarkable change in cytoskeleton, we can see actin condensation near the plasma membrane, and ruffling structure around the membrane. Cytoskeleton of mutant bacteria infected HBMEC varies with respect to each mutant the cytoskeletal change of E44: â–³ibeA, infected HBMEC is almost the same as that of HBMEC infected by E44; the cytoskeletal change of E44: â–³ibeC infected HBMEC is significant but smaller than that of HBMEC infected by E44; The cytoskeletal change of E44: AibeB infected HBMEC can hardly be seen, it is almost the same as negative control HBMEC.(2) E44 infected HBMEC showed remarkable change in cell tight junction. the ZO - 1 stain showed uncontinuous fluorescence on the edge of cell. HBMEC infected by E44: â–³ibeA is almost the same as that of HBMEC infected by E44; there are almost no change of cell tight junction of HBMEC infected by E44:â–³ibeB; E44:â–³ibeC they seemed almost the same as negative control HBMEC.4. There are significant change in cytoskeleton and tight junction of HBMEC stably transfected by ibeB gene containing plasmid P^cHiSC-ibeBincomparison...