Date of Award

8-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

C. Neal Stewart

Committee Members

Timothy J. Tschaplinski, Alison Buchan, Cynthia B. Peterson

Abstract

The increasing prevalence of drug-resistant pathogens is an urgent problem that requires novel methods of bacterial control. Plant extracts inhibit bacterial pathogens and could contain antibacterial compounds with novel mechanisms of action. Yerba mate, a common South American beverage made from Ilex paraguariensis, has antibiotic activity against a broad range of bacterial pathogens. In this work, an attempt was first made to characterize the antibacterial source of an aqueous yerba mate extract by generating a series of extract fractions, collecting GC-MS and antibacterial activity profiles, and then ranking the hundreds of compounds by their presence in fractions with high antibacterial activity. Quinic acid, quercetin, and 5-hydroxy pipecolic acid were highly ranked, suggesting an association between the antibacterial activity of yerba mate against methicillin-resistant Staphylococcus aureus (MRSA). Next, metabolites that accumulated in the supernatants of Salmonella Typhimurium and Lactobacillus casei cultures were surveyed for decreases in phenolic compounds that might signify metabolism of bioactive yerba mate components. No decreases in phenolic compounds were observed. The hypothesis that phenolic compounds might chelate iron as a mechanism of antibacterial activity was also tested; exogenous iron sulfate stimulated the partial recovery of S. Typhimurium to the inhibitory effect of yerba mate in a milk system. Finally, an assessment of potential antibacterial mechanisms of action was undertaken by surveying the metabolites produced by Salmonella Typhimurium in the presence of yerba mate extract and conducting assays to assess cell membrane integrity and catalase activity. No effect on the cell membrane was observed while catalase activity was reduced in the presence of yerba mate extract. Metabolomics revealed significant differences in central carbon metabolism, the cell wall precursor UDP-N-acetylglucosamine, the regulatory metabolites alpha-ketoglutarate and acetylphosphate, the energy metabolite NAD+, and a match to yohimbine, which has known antibacterial activity. Future work can move closer to understanding the antibacterial value of yerba mate extract and its constituents by testing specific mechanistic hypotheses based on metabolic alterations, further examining 5-hydroxy pipecolic acid and yohimbine for antibacterial activity and mechanism, and annotating currently unknown compounds that could have antibacterial activity or be additional key metabolites pointing to specific mechanisms of action.

Table_1_review_summary.pdf (437 kB)
Table 1. Known antibacterial mechanisms of action of plant phenolic compounds.

Table4_top10results.pdf (101 kB)
Table S 4. Top ten results for each attribute ranking method.

Table7_p_ranks_Lcasei_sig.xls (36 kB)
Table 7. L. casei metabolites found to be significantly different between treatments with ‘control_no_bacteria= milk+tea, ‘control_no_tea ’ = milk+bacteria, and ‘tea’ = milk+tea+bacteria. Extracted ion intensity values normalized to a gallic acid internal standard are listed with data ranges in parentheses.

Table8_p_ranks_STyphim_sig.xls (27 kB)
Table 8. S. Typhimurium metabolites found to be significantly different between treatments with ‘control_no_bacteria= milk+tea, ‘control_no_tea ’ = milk+bacteria, and ‘tea’ = milk+tea+bacteria. Extracted ion intensity values normalized to a gallic acid internal standard are listed with data ranges in parentheses.

TableS12_all-untargeted.xls (3350 kB)
Table S 12. Complete list of all metabolites found with LC-MS.

TableS13_all-targeted.xls (81 kB)
Table S 13. List of all identified metabolites found with LC-MS.

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