Submitted to: BMC Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/10/2014
Publication Date: 2/21/2014
Publication URL: http://handle.nal.usda.gov/10113/59367
Citation: Kudva, I.T., Stanton, T.B., Lippolis, J.D. 2014. The Escherichia coli O157:H7 bovine rumen fluid proteome reflects adaptive bacterial responses. BMC Microbiology. 14:48. DOI: 10.1186/1471-2180-14-48. Interpretive Summary: Cattle are the primary source of Escherichia coli O157 (O157), a bacteria (micro-organism) responsible for bloody diarrhea and other serious symptoms in humans that can even lead to death. Although O157 can cause serious disease in humans, cattle infected with this bacteria remain disease free. The first compartment of the cattle gastrointestinal (digestive) tract (GIT) that O157 enters in to after being eaten along with feed is the rumen, where digestion of food begins. O157 needs to adapt to the environment in the rumen which comprises of regurgitated and partially digested food particles and rumen fluid (RF), in order to survive and successfully pass through to the other compartments of the cattle GIT before settling in its actual colonization site close to the end of this GIT. Identifying O157 proteins that are produced by this bacteria during this process of adaptation in the rumen could give us a better understanding of the mechanisms used by these bacteria to infect cattle which in turn, may be used to develop methods to block and remove O157 from these animals. Reducing or removing O157 from cattle would decrease the possibility of this bacteria infecting humans. We decided to address this issue by growing O157 in RF, and identifying O157 proteins that are produced in RF, under varying conditions as seen in the actual bovine rumen, using cutting-edge proteomics technology. We have identified O157 proteins that are specifically produced in the RF and are presently verifying/confirming their role in O157 survival. This information could then be used to develop methods to effectively block O157 colonization of cattle.
Technical Abstract: We defined the growth characterisitcs and proteome of Escherichia coli O157:H7 (O157) cultured in rumen fluid (RF; pH 6.0 -7.2 and low volatile fatty acid content) obtained from rumen-fistulated cattle fed low protein content “maintenance diet”under diverse in vitro conditions to obtain insights into O157 survival mechanisms in the bovine rumen. Bottom-up proteomics (LC-MS/MS) of whole cell-lysates of O157 cultured under anaerobic conditions in filter-sterilized RF (fRF; devoid of normal ruminal microbiota) and nutrient-depleted and filtered RF (dRF) resulted in an anaerobic O157 fRF- and dRF-proteome comprising 35 proteins functionally associated with cell structure, motility, transport, metabolism and regulation, but interestingly, not O157 virulence. Shotgun proteomics-based analysis using isobaric tags for relative and absolute quantitation was used to further study differential protein expression in unfiltered RF (uRF; RF containing normal rumen microbial flora) complemented these results and futher indicated that in the rumen, the first anatomical compartment encountered by this human pathogen within the cattle gastrointestinal tract (GIT), O157 initiates a program of specific gene expression that enables it to adapt to the in vivo environment, and successfully transit to its colonization sites in the bovine GIT. Further experiments, in vitro using uRF from animals fed different diets and with additional O157 strains, and in vivo using rumen-fistulated cattle will provide a comprehensive understanding of the adaptive mechanisms involved, and help direct evolution of novel modalities for blocking O157 infection of cattle.