Application of nuclear magnetic resonance based metabolomics to study the central metabolism of staphylococci

Metabolomics studies the collection of small molecules (metabolites) involved in enzymatically catalyzed reactions, cell signaling and cellular structure. Perturbations in metabolite concentrations have been used to reflect the activity of corresponding enzymes or proteins. Nuclear magnetic resonance (NMR) spectroscopy is a well-known approach for the structure determination of biological macromolecules. Alternatively, NMR has recently been established as a valuable tool of metabolomics, in which NMR spectral signals correlate small molecules with cellular activities. This has been accomplished through the chemometric analysis of high-throughput one dimensional 1H spectra (metabolic fingerprinting) and quantitative metabolite identification based on two dimensional 1H, 13C HSQC spectra (metabolic profiling). Staphylococcus aureus and Staphylococcus epidermidis are the leading pathogens that contribute to a large portion of fatal infections in the USA because of their virulence factors, abilities to survive and thrive on various hosts, and enhanced drug-resistance through biofilm formation. The ability of pathogens to sense environmental conditions and to mediate an adaptation of its metabolism to various conditions was studied using NMR-based metabolomics. My research projects included: the correlation between TCA cycle inactivation and biofilm formation related stressors in S. epidermidis; investigating the metabolic details contributing to biofilm formation in S. epidermidis under conditions that repress TCA cycle activity; establishing a correlations between iron depletion and oxygen limitation in S. aureus metabolism; and evaluation of CcpE as a positive TCA cycle regulator in S. aureus. Ribose phosphate isomerase R (RpiR) is a transcriptional regulatory protein involved in the pentose phosphate pathway. Inactivation of the TCA cycle increases carbon flow into the pentose phosphate pathway where the RpiR protein family may be involved. In S. aureus, mutations of intracellular ribose sensing regulators (members of the RpiR family) resulted in changes in the synthesis of virulence factors. The inducer for ribose phosphate isomerase A, B and the mechanism by which RpiRa regulates rpiA, rpiB gene expression remain to be elucidated. The C-terminal domain of RpiRa was predicted to be a sugar isomerase binding protein domain using homology modeling and it was overexpressed and purified using an E. coli pET overrexpression system as a first step towards the structural determination of this protein