Zamboni Lab, 13C metabolic flux analysis in Mycobacteria

Goal

Identify intracellular flux profiles of mycobacteria when grown on combinations of carbon sources that are relevant for pathogenesis (e.g. glucose and acetate, glucose and cholesterol derivatives) to work towards mechanisms of flux regulation. 

Background

Mycobacterium tuberculosis, the causative agent of tuberculosis, affects one third of the human population. After air-born infection, mycobacteria are encapsulated by alveolar macrophages, where they manage to survive the immune response and adapt to a limited availability of nutrients. Despite the importance of understanding mycobacterial metabolic adaptations for developing effective anti-tuberculosis therapies, metabolic fluxes in mycobacteria have yet been poorly described.

We use Mycobacterium smegmatis as a model organism to study mycobacterial flux rearrangements during growth on multiple substrates. It has been shown that mycobacteria co-utilize glycolytic and gluconeogenic carbon sources and optimize their flux distributions to increase growth and metabolic robustness. We aim at estimating and analyzing flux rearrangements under such multi-substrate conditions to achieve a better understanding of mycobacterial metabolic plasticity.

Project description

First, M. smegmatis will be grown on single carbon sources and on combinations of those (glucose and acetate, glucose and propionate, propionate and acetate) and the physiological parameters (growth and uptake rates) will be measured to quantitatively assess the benefits of co-metabolism. Second, ¹³C-labeling experiments with different labeling strategies will be performed to estimate metabolic fluxes with the standard isotope balancing method and flux ratio analysis methods developed in the lab. Third, mutant strains lacking enzymes involved in anaplerosis via the glyoxylate shunt will be used to challenge the hypothesis of advantageous co-metabolism. Overall, the project should describe metabolic fluxes in mycobacteria co-metabolizing multiple substrates and hence provide a better understanding of the mechanisms of flux regulation in these bacteria.

Methods

Basic microbiology protocols, ¹³C-labeling techniques, liquid chromatography, metabolite mass-spectrometry measurements (LC-MS/MS, GC-MS), stationary ¹³C flux analysis (global isotope balancing, flux ratio analysis).

Requirements

The candidate should have a strong interest in doing basic research, learning new experimental and data analysis techniques and be ready to perform scientific communications in English. No prior knowledge is required, however, either wet lab experience or basic programming skills (MatLab, anything else) would be advantageous.

Contact: Maria Kogadeeva