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United States Department of Agriculture

Agricultural Research Service

Research Project: PREVENTION AND CHARACTERIZATION OF PERSISTENT COLONIZATION BY E. COLI O157:H7 AND OTHER SHIGA TOXIN-PRODUCING E. COLI (STEC) IN CATTLE Title: Proteomic analysis of Escherichia coli O157 cultured in bovine rumen fluid

Authors
item Kudva, Indira
item Stanton, Thaddeus
item Lippolis, John

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 20, 2013
Publication Date: May 10, 2013
Citation: Kudva, I.T., Stanton, T.B., Lippolis, J.D. 2013. Proteomic analysis of Escherichia coli O157 cultured in bovine rumen fluid [abstract]. 113th General Meeting, American Society for Microbiology. p. 190.

Technical Abstract: To obtain insights into Escherichia coli O157 (O157) adaptation and survival in the bovine rumen, the first anatomical compartment encountered by this pathogen during transit through the bovine gastrointestinal tract to sites of colonization, we defined the proteome of O157 cultured in rumen fluid (RF) under different growth conditions, in vitro. RF obtained from rumen-fistulated cattle fed a low protein content “maintenance diet” (resulting in RF of pH ranging from 6.5-7.2, and low Volatile Fatty Acid [VFA] content) was used in the study. Whole cell-lysates of O157 cultured under anaerobic conditions in filter-sterilized RF (fRF; devoid of normal rumen microbial flora), were subjected to top-down proteomics (Ge-LC/MS/MS). Results revealed that the proteome of O157 cultured in fRF anaerobically comprised 36 proteins. Identified proteins were of diverse functional classes, and included those associated with cell structure, motility, transport, metabolism and regulation, but interestingly, did not include those contributing to O157 virulence. Twelve proteins were unique to the O157 fRF-anaerobic proteome, and were not part of proteomes of O157 cultured anaerobically in fRF depleted of nutrients (dRF), or the Luria-Bertani (LB) broth; 10/12 proteins were not components of proteomes of O157 grown aerobically in fRF, dRF or LB broth. Based on these results, and to more accurately determine O157 protein expression in the bovine rumen, we are currently studying the anaerobic proteome of O157 cultured in unfiltered RF (uRF), and employing comparative proteomics-based approaches (iTRAQ) to earmark proteins that are differentially expressed in the anaerobic O157 fRF and uRF proteomes for further evaluation in cattle. We anticipate these studies to facilitate a better understanding of adaptive mechanisms employed by this human gastrointestinal pathogen to adapt to, counter and survive in the hostile rumen environment. A comprehensive understanding of such mechanisms could eventually lead to development of modalities for blocking O157 colonization of bovine reservoirs.

Last Modified: 7/28/2014
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