Exploring in silico Metabolic Phenotypes with a Genome-Scale Model of Mycobacterium Tuberculosis

Tuberculosis (TB) remains as an important health problem throughout the world and was responsible for 1.5 million deaths in 2013 (WHO, 2014). Mycobacterium tuberculosis (Mtb), the agent responsible for human TB, is a facultative intracellular pathogen that infects the human host under a stringent environment that has been associated with Mtb metabolic flexibility. Understanding the different metabolic phenotypes observed during Mtb’s adaptation to different nutritional environments is useful for generating insights about the vulnerability of the pathogen and TB control.

In this work, a methodology based on sensitivity analysis was followed for exploring in silico different metabolic phenotypes of Mtb that have been experimentally observed. For this purpose, an extended genome scale model of Mtb was used. The model encompasses 1021 metabolites and 1265 reactions catalyzed by 922 genes modified with β oxidation and cholesterol degradation pathways. Maximization of growth per flux unit was used as objective function in a two-step optimization strategy. For the first stage, Flux Balance Analysis (FBA) was used for maximizing growth rate (primal LP problem). The solution of the dual LP problem allowed us to calculate the shadow prices for each metabolite and the construction of phenotypic phase plane diagrams in order to explore metabolic phenotypes at different oxygen and carbon source availability. The second stage dealt with the minimization of the squared sum of all fluxes (non-linear programming problem) of the metabolic network model and was used to calculate a unique flux distribution in each phenotype (in order to deal with the problem of the existence of alternate optimal solutions). Two case studies were analyzed. Case study I addressed the analysis of phenotypes derived from the variation of glucose uptake and oxygen consumption in a ΔpfkA mutant. Case study II deals with the analysis of phenotypes derived from the variation of propionate uptake and oxygen consumption in a Δicl mutant.

Obtained in silico results confirm  some hypothesis already observed experimentally by Muñoz-Elías et al., (2006); Savvi et al., (2008) and Lee et al., (2013). Such hypothesis are related to the use of glycolysis, TCA cycle and propionate metabolism. In silico observations showed us that during adaptation to hypoxia, Mtb slows and redirects its TCA cycle to increase production of succinate by fumarate reductase while sustaining menaquinone pool and ATP synthesis. This phenotype has a detrimental effect for growth when glucose uptake increase in a ΔpfkA mutant, first by the generation of toxic sugar phosphate intermediates and second by the increase of carbon flux towards the reductive direction of the TCA cycle. Additionally, we show the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA during the growth of Mtb at low propionate uptake rates. At high levels of propionate uptake, we found a decrease in the TCA cycle intermediates which produces an imbalance either in NADH/NADPH or FADH₂ and leads to low ATP synthesis. Our results offer new insights about the optimal use of Mtb metabolic pathways during exigent nutritional environmental conditions, and shows the advantages of using genome-scale models in Tuberculosis research.

References

Lee, W., VanderVen, B. C., Fahey, R. J., & Russell, D. G. (2013). Intracellular Mycobacterium tuberculosis exploits host-derived fatty acids to limit metabolic stress. Journal of Biological Chemistry, 288(10), 6788-6800.

Muñoz‐Elías, E. J., Upton, A. M., Cherian, J., & McKinney, J. D. (2006). Role of the methylcitrate cycle in Mycobacterium tuberculosis metabolism, intracellular growth, and virulence. Molecular microbiology, 60(5), 1109-1122.

Savvi, S., Warner, D. F., Kana, B. D., McKinney, J. D., Mizrahi, V., & Dawes, S. S. (2008). Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids. Journal of bacteriology, 190(11), 3886-3895.

WHO. (2014). Global Tuberculosis Report 2014. World Health Organization.