Metabolomic Analysis of Escherichia coli O157:H7 Under Acid Stress Using Two-dimensional Gas Chromatography-time-of-flight Mass Spectrometry.

FLETCHER, KELLY ANN. Metabolomic Analysis of Escherichia coli O157:H7 under Acid Stress using Two-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry. (Under the direction of Suzanne Johanningsmeier and David Muddiman). Escherichia coli O157:H7 is a human pathogen noted for its ability to resist acidic environments. The acid resistance of this serotype is understood through two hypotheses. Organic acids that cross the cell membrane of E. coli at pH 3.2 decrease the intracellular pH upon dissociating inside; to counteract this, the cell expends ATP to run the primary proton pump in reverse of the proton motive force and transport protons out of the cytoplasm. E. coli also has four acid resistance (AR) mechanisms, three of which rely on the decarboxylation of amino acids. Researchers have presented genomic, transcriptomic, and proteomic evidence to support these mechanisms in Escherichia coli O157:H7, but a metabolomic approach has not yet been used to study acid resistance in this serotype. The objective of this work was to use a GCxGC-ToFMS metabolomic platform to discover the metabolic changes in E. coli O157:H7 under organic acid and acid pH stress. A microbial metabolomics experiment was designed to test the properties of an acidresistant strain of E. coli O157:H7, B241 (28RC1). Cells were exposed to acid pH (3.2) in the presence and absence of acetic acid, a commonly used food acidulant, in order to induce changes in the intracellular metabolite pool. Cells in identical buffer at unstressed pH (7) were used as a control. After acid-stressing the cells, they were filtered from the treatment medium and quenched. Metabolites were extracted and chemically derivatized for analysis by two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-ToF-MS). Work was undertaken to ensure the conditions used in the experiment were sufficient to induce acid stress without resulting in cell death. It was found that strain B241 survived acid pH conditions with and without 40 mM acetic acid as an additional stressor, at 99 % survival over a thirty minute exposure, eliminating the concern of detection of metabolites from dead cells. The quantity of cells to be treated, collected, and analyzed in order to detect cellular metabolites was optimized. Experimental measurement of cell dry weights and the visual analysis of chromatograms indicated that the ideal amount of cells for this work was equivalent to approximately 3 mg dry weight. Derivatization and GCxGC-ToF-MS analysis of pure standard compounds was conducted to confirm the identities of metabolites. Furthermore, measurement of time and temperature conditions for the halting of cellular metabolism (“quenching”) was performed from which a method was selected which quenched the cells at -40 °C in 9.33 ± 2.50 seconds. An experiment was carried out which compared the intracellular metabolite pools of E. coli O157:H7 which had been exposed to neutral pH, acid pH, or acid pH plus 40 mM acetic acid for thirty minutes. Seven independent biological replicates were measured and sample order was randomized. Acid pH decreased the concentration of glutamate and increased the concentration of GABA, supporting current theories explaining acid resistance in O157:H7. Hierarchical clustering analysis uncovered 27 metabolites that decreased significantly (including amino acids and nitrogenous metabolites) and 20 that increased significantly, solely in response to acetic acid stress (p < 0.001). These results represent a promising first look into the metabolomics of acid-stressed E. coli O157:H7, which directly contributes to enhanced knowledge about the acid resistance properties of this serotype. © Copyright 2012 by Kelly Ann Fletcher All Rights Reserved Metabolomic Analysis of Escherichia coli O157:H7 under Acid Stress using Two-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry by Kelly Ann Fletcher A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science