Metabolomics and Proteomics of Bacterial Signaling
EMSL Project ID
30480
Abstract
The purpose of this work is to understand the role of the microbial methionine salvage pathway specific enzyme Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTN) in bacterial autoinducer signaling and pathogenesis by studying the effects of genetic and pharmacologic inhibition of MTN activity. Due to the role of MTN in regulating the levels of MTA and SAH, potent inhibitors of cellular growth and precursors for bacterial signaling molecules, interruption of MTN activity should lead to significant attenuation of microbial processes. The proteomic and metabolomic study of bacterial adaptations to MTN deficiency may highlight metabolic responses that are amenable to drug intervention and reveal mechanisms by which drug resistance is mediated. Armed with this information we will be better able to design new and better drugs and attenuated vaccines for bacterial pathogens. Prior work in the Cornell lab has focused on the cloning, expression and kinetic study of target bacterial MTNs, and the testing of analogs and transition state compounds for enzyme inhibitory and antimicrobial activity. Analysis of E. coli cultures treated with MTN inhibitors or containing genetic deletions in MTN reveal significant delays in growth and reduced biofilm formation. The results of these studies lead us to the following hypothesis: Interruption of MTN activity will lead to nutrient and autoinducer deficiencies causing global changes in cellular protein and metabolite constituents. Changes to these characteristics could be responsible for attenuation of bacterial virulence, and alterations to drug resistance phenotypes. To investigate this hypothesis, we will use E. coli and Klebsiella pneumoniae model systems since these represent significant pathogens for which the most study of MTN and methionine salvage have been performed. As well, the E. coli proteomic and metabolomic profiles have been established in response to other environmental and genetic manipulations, so the results of our study will have ready comparisons in the scientific literature and contribute significantly to highly cited publications in the area of bacterial quorum sensing. The metabolomics and proteomics of this bacterial signaling pathway will be studied by Drs. Cornell and McDougal using a combination of traditional biochemical characterization methods (2D Gel electrophoresis, RT-PCR, HPLC, spectrophotometry, etc.) in addition to NMR spectroscopy and LC-MS-MS. Initial studies will focus on the identification and quantitation of proteins and metabolites that are most likely to respond to alterations in MTN activity (i.e. those involved in methionine, purine and carbohydrate metabolism). Subsequent studies will examine the global effects on protein expression and metabolite levels that may accompany the interruption of MTN dependent autoinducer signaling events. Analysis on the EMSL LTQ Orbitrap-MS and FT-ICR-MS will improve the accuracy and breadth of the proteomic evaluation of bacterial MTN knock-out strains that are being conducted by low throughput 2D Gel electrophoresis / LC-MS at BSU. Access to the 600 MHz NB Varian Inova-LC-NMR system equipped with a metabolomics cryoprobe, Chenomx metabolomics spectrum deconvolution software, state-of-the-art mass spectrometers, and Senior Research Scientists at PNNL-EMSL will substantially facilitate progress on the proposed research.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2008-09-05
End Date
2010-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members