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United States Department of Agriculture

Agricultural Research Service

Research Project: PREVENTION OF LOSSES FROM COLIBACILLOSIS AND O157:H7 AND OTHER SHIGA TOXIN-PRODUCING E. COLI (STEC) IN CATTLE AND SWINE Title: Proteomic analysis of Escherichia coli O157 for discovery of novel adhesins

Authors
item KUDVA, INDIRA
item Griffin, Robert -
item Krastins, Bryan -
item Sarracino, David -
item Calderwood, Stephen -
item Manohar, John -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 28, 2011
Publication Date: May 23, 2011
Citation: Kudva, I.T., Griffin, R.W., Krastins, B., Sarracino, D.A., Calderwood, S.B., Manohar, J. 2011. Proteomic analysis of Escherichia coli O157 for discovery of novel adhesins. Meeting Abstract. Paper No. 1024.

Technical Abstract: Background: Cattle are primary reservoirs of the food borne human pathogen Escherichia coli O157 (O157). Given that the complement of factors contributing to O157 adherence to epithelial cells at the recto-anal junction and other intermittent anatomical sites along the bovine gastrointestinal tract (GIT) is yet to be elucidated, our goals here were to (i) define the proteome of O157 grown under in vitro conditions that mimic the in vivo host environment using a proteomics-based approach; and (ii) identify novel adhesins by evaluating a selected subset of O157 proteome in a new in vitro bovine-cell adherence assay. Methods: O157 was cultured in LB broth and DMEM with and without norepinephrine (NE) to an OD 600 of 1.0. Bacterial cells were harvested, lysed, and proteins in lysate and pellet fractions subjected to one dimensional SDS-PAGE liquid chromatography tandem mass spectrometry (GeLC-MS/MS). Protein identification was by SEQUEST database searching of the O157 genome databases. Hypothetical/unknown proteins were characterized using bioinformatics. Following comparative analyses of O157 LB±NE and DMEM±NE proteomes, genes encoding proteins of interest were cloned into a non-pathogenic E. coli strain and evaluated for their ability to confer adherence to this strain in a new bovine cell adherence assay. Results: The DMEM+NE O157 proteome (n=704 proteins) was constituted by proteins of diverse functional classes that localized to all cellular compartments, and mapped to the backbone as well as to “O” islands sequences. This proteome included several previously identified O157 virulence factors, including (i) proteins comprising the O157 immunome in both cattle reservoirs and humans reported by us in previous studies; (ii) orthologs of adhesins in distantly related pathogens; and (iii) unknown/hypothetical secreted and outer membrane proteins. Of these, select proteins are currently under evaluation for their adherence potential. Conclusion: The present study resulted in the identification of a panel of interesting proteins that could contribute to adherence of O157 to the bovine GIT. The study also highlights an assay for directly evaluating adherence of pathogen proteins to bovine GIT epithelial cells.

Last Modified: 9/29/2014
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