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We hypothesize that the unique EHEC effector proteins are responsible for the resulting clinical sequelae, since they are primarily responsible for manipulating intestinal host-cell biology. We suggest a novel, mechanistic idea for exploration in this study, i.e. that the EHEC type III secretion system functions efficiently to let more Shiga toxins enter the host cells because it widens para-cellular junctions to expose more toxin receptors. In order to test this hypothesis, the project will involve the detailed molecular study and comparison of eight different EHEC strains. The three major goals are to: sequence the EHEC locus of enterocyte effacement (pathogenicity island), comparison of the secreted proteomes of these strains, and comparison of host cell signaling alterations induced by these strains.
<p>NON-TECHNICAL SUMMARY:<br/> Shiga-toxin producing Escherichia coli (STEC) are pathogens responsible for outbreaks of food-borne infections. A subset of STEC also harbor a pathogenicity island (locus of enterocyte effacement or LEE) that encodes a type III secretion system; EHEC 0157:H7 is a prominent strain within this sub-group. EHEC infections can lead to watery or bloody diarrhea and, in severe cases, to disseminated disease including hemolytic uremic syndrome and death. Of the 265,000 infections caused annually in the US by STEC, 36% are caused by EHEC 0157:H7 (CDC). Ruminants, especially cattle, are often the direct or indirect source of human STEC infections, with prevalence in feces ranging from 0.2% - 48.8% in the US. My research focuses on the interaction of pathogenic Escherichia coli with host intestinal epithelial cells; we use traditional as well as
cutting-edge proteomic and genomic approaches to understand how this interaction contributes to disease. For the current study, we seek to understand if there is a common set of features shared by EHEC strains that have been responsible for past outbreaks.
<p>APPROACH:
<br/>EHEC genetic island sequencing We will sequence the entire genetic island called the locus of enterocyte effacement (LEE) of 13 outbreak strains of EHEC (Table 1). This region contains genes encoding the type III secretion system and its effectors. Parallel, deep sequencing will be performed, and sequences analyzed for important differences and similarities. We will specifically assess unique signatures in the Type III secretion needle and effector proteins from the EHEC strains, as compared to known signatures in EPEC. 2. Proteomic analyses of EHEC secretomes We will perform fully-automated, comparative iTRAQ analysis of the EHEC secretome, i.e. all the proteins secreted by the outbreak strains, including, but not limited to, the effector molecules. Variations in the number, type, and abundance of molecules that are secreted by the strains will be noted,
since they will inform the studies proposed n the final objective. We have significant experience with these cutting-edge analyses, and they represent a significant strength of our laboratory. 3. EHEC effects on human intestinal epithelial cell biology We will define pathogen-host interactions by studying how EHEC outbreak strains manipulate intestinal host-cell biology. These efforts are a focus of other projects ongoing in the laboratory (no overlap with this work) and represent a unique molecular strength of our research group. For these studies, we will infect human intestinal epithelial cells as a surrogate for infection of the human intestine. Two important outcomes will be evaluated. First, we will measure the efficiency of bacterial adherence to host-cells and the disruption of the tight junctions between the cells. The efficiency of adherence will be measured through an adhesion
assay that we have optimized in the laboratory for EHEC, and para-cellular junction disruption will be measured by testing the electrical resistance of host-cell monolayers. This trans-epithelial resistance (TER) is a sensitive measure of the para-cellular integrity; if the tight junctions between host cells are disrupted, less resistance is offered to the passage of an electric current. If host cells are tightly juxtaposed, then the electrical resistance is greater. TER studies will be validated via immunofluorescence assays, directly visualizing the specific para-cellular junctional proteins ZO-1 and occludin following EHEC infection. We are well-versed in performing these studies, and the laboratory is equipped with all the instrumentation necessary for their success. These tests will show how the bacterial strains behave and affect the human tissue culture cells. The overarching goal
of this project is to correlate genome and proteome variations in EHEC, and to mechanistically define the impact of such variations. Secreted effectors in general represent unique targets to combat bacterial infections in an era of traditional antibiotic resistance, and such information is even more urgently needed for EHEC since antimicrobials are contra-indicated.</p>