Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/31/2018
Publication Date: N/A
Technical Abstract: Introduction. Cattle are the primary E. coli O157 (O157) reservoir and principal source of human infection. The anatomical site of O157 persistence is the bovine recto-anal (RAJ) junction; hence, an in-depth understanding of O157-RAJ interactions will help develop novel modalities to limit O157 in cattle. Towards this goal, thus far, we have (i) analyzed O157 adherence at a histological level; (ii) developed a novel adherence assay using squamous epithelial cells at the RAJ; (iii) evaluated the role of well-characterized STEC adherence proteins in STEC attachment to the RAJ; (iv) explored similarities in RAJ-STEC interactions with another ruminant animal; and (v) employed systems based approaches to prioritize a subset of pathogen proteins likely to play a role in adherence. In this study, we report on bacterial and host proteins constituting the pathogen-RAJ-cell interactome. Material and Methods. O157 ligands: RSE and FAE cells constituting the RAJ were extracted from necropsy tissue samples of uninfected animals. Cell surfomes were surface-biotinylated and used to prepare a “cell-column” under non-reducing conditions. O157 proteins from overnight cultures grown in DMEM or DMEM with norepinephrine (DMEM-NE) were interacted with the cell-column; bound proteins were eluted and analyzed via bottom-up proteomics. To confirm their role in O157 adherence to RSE/FAE cells, adherence-inhibition assays were set up with the eluted proteins after filter-concentration, using standard protocols. A quantitative determination of average numbers of bacteria adhering to RAJ cells was made and statistically evaluated for significance (p less than 0.05). RSE/FAE cells without exposure to concentrated proteins served as controls. RAJ receptors: Eluted O157 proteins were covalently linked to columns to generate “bacterial-column”. RSE/FAE cell surfome was applied to the bacterial-column. RAJ proteins interacting with the bacterial ligands were eluted, and are presently being analyzed as above. Results and Conclusion. Eluted O157 proteins successfully blocked adherence of O157 to RSE and FAE cells. We identified a total of 84 and 89 proteins comprising the RSE and FAE interactome, respectively. Of the identified proteins those with a higher threshold cut off, and most likely to be involved in adherence are being selected for purification. Peptide analogs libraries for application as anti-adherence therapeutics for O157 are being developed.