The Discovery And Verification Of The Interaction Between ZBED1in Host Cells And NleL From Enterohemorrhagic Escherichia Coli (EHEC)O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7is an importantcause of food-and water-borne illnesses in the world. It can deliver effectorsinto eukaryotic cells through a type III secretion system (TTSS) and producecharacteristic ‘attaching and effacing’(A/E) lesions. Its infections can causehemorrhagic colitis (HC) and can result in potentially fatal hemolytic uremicsyndrome (HUS). Thus it is a kind of pathogen that attracts the world-wildattention. One of its virulence-related protein secreted by TTSS, NleL(non-lee-encoded-effector-Ligase), was found to be an E3uniquitin ligase,but its substrates and functional mechanisms in eukaryotic cells were stillunclear. Construction of recombinant, protein induction and purification,protein cellular location, yeast-two hybrid technique (YHT), GST pull-downassays and in-vitro ubiquination assays were used to screen and verify thepotential protein substrates of NleL in eukaryotic cells in this study, weinvestigated the effector protein NleL from the following aspects：1. Potential protein substrate, ZBED1, interacting with NleL in the hostscreened through yeast-two hybrid techniqueZBED1, a eukaryotic cell protein, which might interact with effectorNleL was first screened through yeast-two hybrid experiments.Positiveclones growing in SD-2and SD-4plates were verified as ZBED1nucliotide sequence after amplification in Ecoli DH5α, double restrictionenzyme digestion, nucliotide sequencing and blast analysis. Therefore.itcould be preliminary inferred that the effector NleL might have interactionswith the protein ZBED1in host cells.2. Induced expression and purification of NleL(GST-NleL) protein In this study, we succeeded in purifying full-length NleL protein,GST-NleL-His,which was attached with double lables, GST-tag and His-tagand its fragment, GST-NleL170-782-WTwith only His-tag. The results showedthat the purification methods with GST-beads were better than that with Ni–NTA. The purified NleL fusion protein also met the requirements of thepurity and quality for further experiments, and it could be stored forsubsequent in vitro experiments.3. Test experiments of combination between the effector NleL and ZBED1proteinGST pulldown assays were conducted to further verify the interactionbetween the effector NleL and ZBED1.Western blot showed that full-lenthprotein,GST-NleL-His, and its fragment, GST-NleL170-782WT, could bothcombine with ZBED1in vitro. Since the fragment had the same interactionwith ZBED1as the full-length protein, it could be speculated that thecombining sites in NleL possibly lied in782-782amino acid residues.4. In vitro ubiquitination assays with the effector NleL and ZBED1Some studies reported that NleL was an E3ligase with a cysteineresidue as its functional site which was the753th residue from the N-terminal.Ubiquitination experiments in vitro with the effector NleL and proteinZBED1were aimed to explore whether the effector NleL played its role onZBED1as an E3ligase. The results showed that ZBED1performed self-ubiquitination in vitro. while the smears were obviously weakened whenprotein ZBED1interacted with full-length protein,GST–NleL, and itsmutant fragment protein, GST-NleL170-782C753A. It could be concluded that theeffector NleL probably influenced the physiological activities of the host cellthrough weakening the self-ubiquitination of ZBED1. The results showedthat the mutant fragment protein, GST-NleL170-782C753Ahad the same functionas the full-length protein, suggesting that the effector NleL functioned assomething else rather than as an E3ligase.5. Expression and localization of NleL protein in eukaryotic cellsTo explore the mechanisms of effector NleL from EHEC O157in eukaryotic cells, it was necessary to know its localization in cells.In thisstudy we successfully constructed a recombinant plasmid pCDNA3.0-UTR-EGFP-NleL-HA which could be expressed as EGFP-NleL fusionprotein in eukaryotic cells.Two cell lines,293T and Hela, were transfectedwith the plasmid and its control plasmid pCDNA3.0-UTR–EGFP-HA. Theobservations indicated NleL protein was mostly scattered within thecytoplasm and a small amount entered into the nucleus; while the control,EGFP fluorescent protein, were distributed in the whole cells obviouslyshowing its non-orientation specificity in the cells. It could be speculated thatthe effector NleL might play different roles while in the cytoplasm andnucleus of eukaryotic cells.In short, it was the first time to discover localization of the effector NleLexpressed in eukaryotic cells. One of potential substrates of NleL in host cells,ZBED1, was successfully screened by yeast-two hybrid techonique. GSTpull-down assays and ubiquitination assaays in vitro were used topreliminarily study the interaction between effector NleL and protein ZBED1.All these would provide clues to the further study to uncover the functionalmechanisms of effector NleL in eukaryotic cells.