Interactions of Pathogenic Escherichia Coli with Host Intestinal Epithelial Cells

NON-TECHNICAL SUMMARY: Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli are related pathogens that cause diarrheal disease in a significant proportion of the population. While EPEC-induced disease is mainly a problem in children in developing countries, EHEC is associated with many outbreaks of food-borne infections in many countries including the United States. A number of recent outbreaks of EHEC infections have been related to contaminated food products, mainly produce items such as spinach. These pathogens share many properties, and an understanding of these shared characteristics will be useful in developing measures to control them. Both bacteria attach to the surface ("epithelial") cells in the intestine. Both pathogens employ a molecular "syringe" to insert specific proteins into the intestinal cells; these proteins change the behavior of the host cells so that they facilitate the survival of the bacteria. One such protein is EspZ; this protein prevents the host cells from dying, thereby allowing the pathogen to survive on the host cells. We study the roles of many of these proteins, including EspZ, in helping the bacteria to set up camp and thereby inducing disease. Understanding these processes is the first step in developing methods to specifically block infections.

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APPROACH: Caco-2 cells will be infected with WT, espZ mutant and complemented strains. Uninfected monolayers will serve as controls. At one hour post-infection, unattached bacteria will be removed, and fresh medium added to the cells. At 1, 2, 4 and 6 hours postinfection, the monolayers will be processed for immunofluorescence assays, or cell extracts will be used for immunoblot analyses. Effect of EspZ on Bcl-2 family members: extracts from infected cells will be immunoblotted with antibodies specific for the Bcl-2 superfamily members. Cytochrome C release: Cell extracts from infected Caco-2 monolayers will be separated into cytosolic and mitochondrial components, followed by immunoblot detection of cytochrome C. Activation of caspase 8:extracts from infected Caco-2 cells will be immunoblotted for total and active caspase 8. Transfection experiments: HeLa cells transfected with plasmid encoded Myc-tagged EspZ will be treated with apoptotic agents that stimulate the intrinsic (staurosporine and etoposide) or extrinsic pathway. Role of flagellin and NF-kappaB activation in limiting apoptosis of host epithelial cells: To explore the role of EPEC flagellin in suppressing apoptosis, Caco-2 monolayers will be infected with WT EPEC, flagellin mutants and corresponding complemented strains. Alternatively, the monolayers will be infected in the presence of specific NF-kappaB inhibitors. Death of infected cells will be evaluated by ethidium homodimer uptake and TUNEL assays. Also, protein extracts from similarly infected Caco-2 cells will be immunoblotted for flagellin, caspase 3, 7 and 9. Mutagenesis approaches: This will help identify residues of EspZ that are important for its pro-survival activities. Specific regions and residues will be targeted for change. The mutagenized constructs will be tested for their ability to complement EPEC deltaespZ and reduce its cytotoxic effects on epithelial cells. Identification of host-cell interacting partner(s) of EspZ: Pull-down studies: Purified EspZHis will be immobilized on a nickel column, and Caco-2 cell extracts passed over the column. After washes, bound proteins will be eluted, concentrated by precipitation, separated by SDSPAGE and stained with Coomassie Blue. Extracts loaded on columns containing only the nickel matrix (but no EspZ) will serve as controls. Bands specific to the EspZ-HeLa column will be excised and submitted for mass spectroscopy. Split ubiquitin yeast two-hybrid system: This system is specifically designed to identify interacting partners of membrane-associated proteins, and is based on the reconstitution of the N- and C-terminal fragments of ubiquitin. Briefly, espZ will be cloned into the bait plasmid pCCW to encode an EspZ-CUB-LexA/VP16 fusion protein (CUB=C-terminal fragment of ubiquitin; LexA/VP16=transcription factor), and transformed into the reporter yeast strain, NMY51. The HeLa cell library cloned into pPR3-C (encoding HeLa cell prey- NUB fusion proteins) will subsequently be transformed into this yeast strain, and selected for growth on medium lacking histidine, and subsequently screened for expressing â-galactoside activity.