Non-labeled quantitative proteomic comparison identifies differences in acid resistance between Escherichia coli O157:H7 curli production variants

Authors: Gunther, Nereus - Jack; Liu, Yanhong; Nunez, Alberto; Paul, Moushumi; Uhlich, Gaylen

Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/16/2013
Publication Date: 1/1/2014
Citation: Gunther, N.W., Liu, Y., Nunez, A., Paul, M., Uhlich, G.A. 2014. Non-labeled quantitative proteomic comparison identifies differences in acid resistance between Escherichia coli O157:H7 curli production variants. Foodborne Pathogens and Disease. 11:30-37.

Interpretive Summary: To appreciate how bacterial food pathogens survive and persist in the environment and on food, we need to be able to understand how they live and the type of proteins they produce in different environments. We have developed a reliable, partially automated, and relatively inexpensive method to identify and quantify the proteins being made by bacteria under specific conditions. We used this technology to compare the proteins being made by two closely related Escherichia coli O157:H7 strains that have previously demonstrated different levels of threat to food safety. From the results of our analysis we were able to see that several proteins responsible for resistance to acidic conditions were made in much greater amounts by one of the bacterial strains compared to another. Additional experiments proved that one of the strains was much more resistant to the acid conditions commonly found in the human stomach. Additionally we were able to show that another previously believed unrelated protein was responsible for keeping the expression of the acid resistance proteins low and making the E. coli strain more susceptible to the conditions commonly found in the human stomach. Since the acidity of the human stomach is a major barrier to disease causing food bacteria; identifying a protein responsible for inhibiting the bacteria’s resistance to this acidic condition is an important discovery.

Technical Abstract: To understand the nature and capability of a bacterial strain to tolerate environmental conditions it is necessary to be able to identify and measure the proteins that they are expressing in any given situation. In this research, the entire protein complements produced by Escherichia coli O157:H7 strain 48394OW and its naturally occurring curli producing variant 48394OR were compared to better understand the capabilities of these two closely related strains. A non-labeled proteomic comparison was performed utilizing the spectra counting and peptide fractionation abilities of a Q-Tof mass spectrometer to identify and quantitate the proteins produced by the two strains. The process reliably identified and measured the concentration of 418 proteins from strains 48394OW and 48394OR within three separate biological replicates. From these two sets,151 proteins were identified that were preferentially expressed in strain 43894OW compared to 48394OR and 68 proteins that were conversely preferentially expressed in 48394OR. A subset of the preferentially expressed proteins was assayed to determine if their levels of gene transcription corresponded with the observed protein expression. From the resulting list of confirmed differentially expressed proteins, it was observed that the proteins contributing to acid survival: GadA, GadB, and GadC were overexpressed in 48394OW compared to 48394OR. These protein predicted an enhanced acid resistant phenotype for 48394OW was confirmed by experimentation.