Determining the Mechanism of Action of MTN-specific Inhibitors by Analyzing Metabolites Using LC/MS
The bacterial specific enzyme 5’-methylthioadenosine/S-adenosylhomocysteine(MTA/SAH) nucleosidase (MTN) is essential for production of autoinducer 2 (AI-2), a secreted quorum sensing signal that influences biofilm formation and virulence in bacteria. To explore this enzyme as a potential target for antibiotic development, we are studying MTN gene deletion strains to determine how loss of MTN activity affects bacterial metabolism. This could help in determining the mechanism of action of MTN-specific inhibitors that are being developed as new antibiotics to treat microbial infections. Metabolomics is the comprehensive and quantitative analysis of metabolites in biological pathways. Our investigation is focused on examining metabolites which are excreted by bacteria during different phases of growth, and which changed in response to MTN gene deletion. The primary goal of this investigation was to create a mass spectral library of standard metabolites using High Pressure Liquid Chromatography (HPLC) and Mass Spectrometry (MS) that could be used to identify the changing metabolite levels in samples of wild-type and MTN knockout E. coli strains. The metabolites we have focused on include those found in central carbon metabolism (pyruvate, lactate, acetate, etc), methionine salvage (methionine, MTA, SAH, etc) and polyamine metabolism (spermidine, ornithine, etc). The results of our analysis indicate that there are unique metabolite profiles that accompany MTN gene deletion, and these suggest that drugs that work on MTN will disrupt S-adenosylmethionine dependent vitamin synthesis.