|GRIFFIN, ROBERT - Massachusetts General Hospital|
|KRASTINS, BRYAN - Thermo Fisher Scientific|
|SARRACINO, DAVID - Thermo Fisher Scientific|
|CALDERWOOD, STEPHEN - Massachusetts General Hospital|
|MANOHAR, JOHN - Pathovacs, Inc|
Submitted to: BMC Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/2012
Publication Date: 6/12/2012
Citation: Kudva, I.T., Griffin, R.W., Krastins, B., Sarracino, D.A., Calderwood, S.B., Manohar, J. 2012. Proteins other than the Locus of Enterocyte Effacement-encoded proteins may contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells. BMC Microbiology. 12(103). Available: http://www.biomedcentral.com/1471-2180/12/103.
Interpretive Summary: Cattle are natural reservoirs of the human pathogen, Escherichia coli O157 (O157). The primary site of O157 colonization in these animals is the recto-anal junction (RAJ) and the proteins encoded by genes on the ‘Locus of Enterocyte Effacement (LEE)’ and secreted by the ‘Type III secretion system (TTSS)’ are considered critical for O157 adherence to the follicle-associated epithelial (FAE) cells at the RAJ. However, we found that these proteins do not appear to have a role in O157 adherence to another type of cells also found at the RAJ, the squamous epithelial (RSE) cells. O157 was cultured under nutrient-limiting conditions of the Dulbecco’s Modified Eagle Medium (DMEM) for the adherence assay, which is reflective of conditions encountered in vivo and also parallels growth conditions used to prepare the commercially available, cattle-use approved, and TTSS protein-based O157 vaccine. Using a proteomics approach, we determined all the proteins that are expressed by O157 during growth in DMEM (DMEM-proteome). This O157 DMEM-proteome comprised of 686 proteins that included several previously characterized as well as uncharacterized proteins, in addition to the TTSS proteins. Experimental evaluation of these additional proteins could help identify a subset of proteins that contribute towards O157 adherence. These would be excellent candidates for developing highly efficacious, anti-adhesion, cattle O157 vaccines as a successful vaccine should be able to eliminate O157 adhering to both the FAE and RSE cells at the RAJ.
Technical Abstract: In this study, the Type III Secretion System (TTSS) proteins considered critical for Escherichia coli O157 (O157) adherence to the follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), did not appear to contribute to O157 adherence to the RAJ squamous epithelial (RSE) cells that also constitute this primary site of O157 colonization in cattle. Hence, to identify proteins that could facilitate adherence of O157 to bovine RSE cells, we defined the proteome of O157 cultured in DMEM (the same growth conditions used to evaluate O157adherence to RSE cells) both to reflect in vivo nutrient-limiting conditions, and to closely parallel those conditions used for the preparation of the cattle-use approved, TTSS-protein based O157 vaccine (Bioniche). The O157 proteome as expressed in DMEM (DMEM-proteome) was analyzed using GeLC-MS/MS, and comprised 686 proteins including, (i) proteins that localized to all cellular compartments, and mapped to the ‘backbone’, as well as the ‘O island’ and ‘pO157’ regions of the O157 genome, (ii) previously identified virulence factors and TTSS proteins, (iii) proteins that comprised the cattle and human O157 immunome as reported by us in previous studies (iv) proteins that were orthologs of adhesins in distantly related pathogens; and (v) numerous unknown/hypothetical secreted and outer membrane proteins. Experimental identification of a subset of proteins in the O157 DMEM-proteome that mediate O157 attachment to bovine RSE cells, and their optimal delivery to the mucosal immune system is likely to result in highly efficacious anti-adhesion vaccines.