Quantitative Mass Spectrometry Reveals Plasticity of Metabolic Networks in Mycobacterium smegmatis

Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.

Metabolic modeling and experimental studies on carbon and energy metabolism of Mycobacterium tuberculosis

Muhittin Emre Özdemir

Tuberculosis is an ancient human disease affecting millions of people every year. The common causative agent, Mycobacterium tuberculosis, is a peculiar pathogen that can persist in the host tissues for decades. Many of the key attributes of M. tuberculosis can be traced to its metabolism. Therefore, novel insights into the metabolism of this bacterium could lead to the identification of new drug targets as well as better methods to understand the mechanism of action of anti-tuberculosis drugs. Analysis of the metabolic network of this bacterium using a genome-scale model offers an unconventional approach to understanding utilization of metabolic pathways of this pathogen during growth and non-replicating persistence. Metabolic adaptation of a pathogen is central to its life style in vivo. To understand the capacity and limitations of M. tuberculosis, we developed an updated and thermodynamically curated genome scale model – iOS783. This model has been verified with respect to the observed phenotypes for targeted gene deletion studies reported previously. Evaluation of iOS783 with thermodynamics based flux balance analysis (TFBA) improved overall reliability of the model results and led to identification of possible bottleneck reactions. In addition, several new hypotheses were generated with iOS783, involving the role of isocitrate lyase enzymes, propionate toxicity, and energy metabolism of this pathogen. Analysis of iOS783 for conditional gene essentiality emphasizes the importance of the carbon source available to the pathogen. Several genes show a conditional impact on biomass production rate of the model, depending on the availability of carbohydrates or fatty acids as the carbon substrate. Conditional essentiality is even more pronounced when ATP replenishment capacity is used as a measure of a pathogen’s ability to survive under stress in a non-growing state. As expected, ATP synthase plays a major role in estimations of ATP replenishment capacity based on iOS783. Curiously, computational analysis of the metabolic network with ATP synthase activity blocked indicates that the damaged network can use substrate-level phosphorylation reactions to sustain ATP production to fulfill non-growth-associated ATP requirements when glucose is available. In order to establish the dependence of M. tuberculosis on ATP synthase, we attempted to construct a deletion of the chromosomal atp operon. Our results show that the native copy of these genes can be deleted only when a second set of genes are introduced elsewhere in the genome, indicating essentiality of the ATP synthase for growth. Although our results indicate that ATP synthase is essential for growth, we observed carbon source specific efficacy of an ATP synthase inhibitor, bedaquiline, on M. tuberculosis. These experimental results are consistent with ATP turnover estimations based on modeling results. Furthermore, analysis of iOS783 suggests a novel role in energy metabolism for recently described glycine dehydrogenase (GDH) on energy metabolism. Deletion of ald gene responsible for GDH activity reduces the ATP replenishment capacity of iOS783, especially during metabolism of fatty acid derivative carbon sources. Consistent with these modeling results, wetlab experiments designed to test this hypothesis show that bedaquiline has a stronger bactericidal effect on an M. tuberculosis ∆ald mutant, compared to the wild-type parental strain, when cells are cultured in medium containing acetate as the carbon source. Metabolic modelling is already well established as an invaluable tool for engineering of strains with desired characteristics. For pathogenic species, however, the application of this approach has so far been quite limited. The work presented in this thesis exemplifies an interdisciplinary approach combining computational modeling and experimental studies of M. tuberculosis metabolism. First, iOS783 was utilized to interpret existing experimental results and to generate novel hypotheses, thereby providing the impetus for new wetlab experiments. Conversely, results from iOS783-inspired wetlab experiments were used to further refine and expand the iOS783 model. This cyclic exchange between computational modeling and wetlab experimentation illustrates how a close interdisciplinary partnership can create a positive feedback loop that enriches both disciplines.

EPFL2013

Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids

John McKinney

Mycobacterium tuberculosis is predicted to subsist on alternative carbon sources during persistence within the human host. Catabolism of odd- and branched-chain fatty acids, branched-chain amino acids, and cholesterol generates propionyl-coenzyme A (CoA) as a terminal, three-carbon (C(3)) product. Propionate constitutes a key precursor in lipid biosynthesis but is toxic if accumulated, potentially implicating its metabolism in M. tuberculosis pathogenesis. In addition to the well-characterized methylcitrate cycle, the M. tuberculosis genome contains a complete methylmalonyl pathway, including a mutAB-encoded methylmalonyl-CoA mutase (MCM) that requires a vitamin B(12)-derived cofactor for activity. Here, we demonstrate the ability of M. tuberculosis to utilize propionate as the sole carbon source in the absence of a functional methylcitrate cycle, provided that vitamin B(12) is supplied exogenously. We show that this ability is dependent on mutAB and, furthermore, that an active methylmalonyl pathway allows the bypass of the glyoxylate cycle during growth on propionate in vitro. Importantly, although the glyoxylate and methylcitrate cycles supported robust growth of M. tuberculosis on the C(17) fatty acid heptadecanoate, growth on valerate (C(5)) was significantly enhanced through vitamin B(12) supplementation. Moreover, both wild-type and methylcitrate cycle mutant strains grew on B(12)-supplemented valerate in the presence of 3-nitropropionate, an inhibitor of the glyoxylate cycle enzyme isocitrate lyase, indicating an anaplerotic role for the methylmalonyl pathway. The demonstrated functionality of MCM reinforces the potential relevance of vitamin B(12) to mycobacterial pathogenesis and suggests that vitamin B(12) availability in vivo might resolve the paradoxical dispensability of the methylcitrate cycle for the growth and persistence of M. tuberculosis in mice.

2008

Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase

John McKinney

Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Despite an urgent need for new therapies targeting persistent bacteria, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of fatty acids. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. A link between the requirement for ICL and the immune status of the host was established by the restored virulence of delta icl bacteria in interferon-gamma knockout mice. This link was apparent at the level of the infected macrophage: Activation of infected macrophages increased expression of ICL, and the delta icl mutant was markedly attenuated for survival in activated but not resting macrophages. These data suggest that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection, an observation with important implications for the treatment of chronic tuberculosis.

2000

Metabolic modeling and experimental studies on carbon and energy metabolism of Mycobacterium tuberculosis

Muhittin Emre Özdemir

EPFL2013

Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids

John McKinney

2008