| PurposePathogens have developed sophisticated mechanisms to either block or subvert normal host cellular processes, thereby inadvertently contributing to patho-genesis and disease outcome. Over the years, a recurring theme in these host -pathogen interactions has emerged: effector molecules of bacteria can mimic host cell molecules to subvert cellular functions, but has also revealed fundamental insights into normal functions of the cytoskeleton. From the use of bacterial toxins to investigate signaling molecules to in vitro studies of actin polymerization u-sing bacteria, the study of pathogenesis has provided important tools to probe the mechanism of pathogenesis and cellular process.Some Gram - negative pathogenic bacteria have acquired sophisticated molecular syringes, such as type III or type IV secretion systems, which are multi-subunit molecular machines that span the bacterial and host membranes and translocate effector molecules directly into host cells. For example, Salmonella directly activate Rho GTPases Cdc42 and Rac using effectors secreted by type III secretion systems, coordinating several signaling pathways that converge to induce transient, actin - rich membrane ruffles that engulf the infecting bacteria. InlB of Listeria could specifically bind the hepatocyte growth factor receptor (HGF - R or Met) , therefore inducing the endocytosis of Listeria into host cells, with concomitant signaling pathway changes. Escherichia coli ( E. coli ) is the common pathogen inducing neonatal meningitis. The neonatal meningitis is mostly produced by bacteremia of E. coli , but the mechanism of E. coli cross-ing brain - blood barrier composed by brain microvascular endothelial cells( BMEC) is still unclear presently. Therefore, elucidating the molecular mechanism involved in E. coli crossing blood - brain barrier may provide targeting point for the prevention of neonatal meningitis.In the process of investigating the mechanism of E. coli crossing blood -brain barrier, we have identified a invasion gene contributing to the pathogenesis of E. coli meningitis, named as ibeB (Invasion of brain endothelial cell). The open reading frame of ibeB is 1383bp, and the molecular weight of putative pro-tein is about 50kD. The preliminary results showed that the outer membrane pro-tein coded by ibeB may be spliced by itself, which point to the similarity of IbeB protein and type secretion of bacteria. Recently, we have observed a new phe-nomenon.- the mouse stem cell transfected with ibeB gene acquire neuronal cell morphological changes, and expression of Nestin gene.Based on our results about ibeB gene, we clone ibeB gene into the eukaryot-ic expression plasmid pcDNA3. 1/HisC, and select human cervical cancer cell line Hela as host for stable transfection. We have observed the biological char-acter changes and made some further analysis about the molecular mechanism of the IbeBs function.Methods1 Construct1. 1 Preparation of plasmid DNA of pcDNA3/7C ( ibeB inserted) and pcD-NA3. 1/HisC, directionally clone ibeB (1. 7kb) into pcDNA3. 1/HisC1. 2 Double digestion to confirm the cloning process, confirm the reading frame of ibeB by sequencing1. 3 Midi - preparation of plasmid DNA for transfection using Qiagen Midi Prep Kit2 Stable transfection2. 1 The expression plasmids were transfected into HeLa cells by Fugene ?6 transfection reagent (Roche) according to the manufacturers instruction. The transfected cells were selected in geneticin for 10 -14 days. The resulting single colonies were multiplied as stable cell lines.2. 2 Western blot analysis of His6 - IbeB fused protein expression using His - tag antibody3 Biological analysis of Hela cells over - expressing IbeB3. 1 Cell morphology changes of living cell photographed under phase - con-trast microscope3.2 Filamentous actin expression displayed by rhodamin - phallodin 3. 3 Cell - substratum adhesion assay 3.4 Cell spreading assay3. 5 Wound healing assay to test the migration rate of Hela cells over - ex-pressing IbeB3.6 Vinculin expre...
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