A human alveolar macrophage genome-scale metabolic reconstruction was reconstructed from tailoring a global human metabolic network, Recon 1, by using computational algorithms and manual curation.A genome-scale host–pathogen network of the human alveolar macrophage and Mycobacterium tuberculosis is presented. This involved integrating two genome-scale network reconstructions.The reaction activity and gene essentiality predictions of the host–pathogen model represent a more accurate depiction of infection.Integration of high-throughput data into a host-pathogen model followed by systems analysis was performed in order to elucidate major metabolic differences under different types of M. tuberculosis infection.
Mycobacterium tuberculosis (M. tb) is an insidious and highly persistent pathogen that affects one-third of the world's population (WHO, 2009). Metabolism is foundational to M. tb's infection ability and the ensuing host–pathogen interactions. In addition, M. tb has a heterogeneous clinical presentation and can infect virtually every tissue. Depending on the location of the infection, different metabolic pathways are active and inactive in both the host and pathogen cells. In this study, we sought to model the host–pathogen interactions of the human alveolar macrophage and M. tb as well as detail the metabolic differences in specific infection types using genome-scale metabolic reconstructions (Figure 4A).
Genome-scale metabolic reconstructions are knowledge bases of all known metabolic reactions of a given organism. Reconstructions have been shown to elucidate the mechanistic genotype-to-phenotype relationship through the integration of high-throughput and physiological data (Oberhardt et al, 2009). Genome-scale reconstructions are converted into mathematical models under the constraints-based reconstruction and analysis (COBRA) platform (Becker et al, 2007). COBRA models use network stoichiometry and steady-state mass balances to define a solution space of potential flux states that a network can take. Thus, the COBRA approach does not require kinetic parameters.
Recently, the global human metabolic network, Recon 1, has been reconstructed (Duarte et al, 2007). To understand the metabolic host–pathogen integrations of M. tb with its human host, we first tailored the global human metabolic network into a cell-specific metabolic reconstruction of the human alveolar macrophage. This was carried out using established computational algorithms (Becker and Palsson, 2008; Shlomi et al, 2008) and manual curation to confirm the included and excluded reactions. The human alveolar macrophage reconstruction, iAB-AMØ-1410, accounts for 1410 genes, 3012 intracellular reactions, and 2572 metabolites (Figure 4C). iAB-AMØ-1410 was able to accurately predict maximum ATP and NO production rates obtained from experimental data (Griscavage et al, 1993; Newsholme et al, 1999).
The second step to studying host–pathogen interactions was integration of the human alveolar macrophage reconstruction with an existing genome-scale metabolic model of M. tb, iNJ661 (Jamshidi and Palsson, 2007). Interfacial constraints were set to create a phagosomal environment that was hypoxic, nitrosative, rich in fatty acids, and poor in carbohydrates. From the onset, it was apparent that some oxygen (<15% of in vitro uptake) was required for proper simulations. In addition, algorithmic tailoring of the M. tb biomass objective function was performed to better represent an infectious state. The integrated host–pathogen metabolic reconstruction was dubbed iAB-AMØ-1410-Mt-661.
Analysis of the integrated host–pathogen metabolic reconstruction resulted in three main findings. First, by setting interfacial constraints and tailoring the biomass objective function, the solution space better represents an infectious state. Without adding artificial constraints to the host portion of the integrated model, the iAB-AMØ-1410 solution space is greatly reduced (Figure 4B). Macrophage glycolysis and nitric oxide production are up-regulated and macrophage ATP production, nucleotide synthesis, and amino-acid metabolism are suppressed. In addition, M. tb glycolysis is suppressed and isocitrate lyase is up-regulated for generation of acetyl-CoA. Fatty acid oxidation pathways and production of mycolic acids are increased, while production of nucleotides, peptidoglycans, and phenolic glycolipids are reduced. The modified solution space of the alveolar macrophage and M. tb better represents the infectious state.
Second, the host-pathogen model more accurately predicts M. tb gene deletion tests than the current in vitro model, iNJ661. The host-pathogen model predicted 11 essential genes and 37 unessential genes differently than iNJ661. A total of 22 of the differentially predicted genes have been experimentally characterized (Sassetti and Rubin, 2003; Sohaskey, 2008). The host-pathogen model correctly predicted 18 of the 22 genes. Thus, iAB-AMØ-1410-Mt-661 is a more accurate platform for studying infectious states of M. tb.
Finally, we sought to determine metabolic differences in both the macrophage and M. tb between three different types of infection: latent, pulmonary, and meningeal. Transcription profiling data of the macrophage for the three infections (Thuong et al, 2008) were integrated in the context of the host–pathogen network to elucidate the reaction activity of the three infections. There was wide heterogeneity in the three infection states; some of these differences are highlighted. Macrophage hyaluronan synthase and export were only active in the pulmonary infection. This is potentially interesting from a pharmaceutical viewpoint as hyaluronan has been implicated as a potential carbon source for extracellular M. tb (Hirayama et al, 2009). In addition, we detected metabolic activity differences in M. tb pathways that have been previously discussed as potential drug targets (Eoh et al, 2007; Boshoff et al, 2008). Polyprenyl metabolic reactions were only active in the latent state infection, while de novo synthesis of nicotinamide cofactors was only active in latent and meningeal M. tb infections.
Host-pathogen modeling represents a novel approach for studying metabolic interactions during infection. iAB-AMØ-1410-Mt-661 is a more accurate platform for understanding the biology and pathophysiology of M. tb infection. Most importantly, genome-scale metabolic reconstructions can act as scaffolds for integrating high-throughput data. Particularly, in this study we were able to discern reaction activity differences between different infection types.
Metabolic coupling of Mycobacterium tuberculosis to its host is foundational to its pathogenesis. Computational genome-scale metabolic models have shown utility in integrating -omic as well as physiologic data for systemic, mechanistic analysis of metabolism. To date, integrative analysis of host–pathogen interactions using in silico mass-balanced, genome-scale models has not been performed. We, therefore, constructed a cell-specific alveolar macrophage model, iAB-AMØ-1410, from the global human metabolic reconstruction, Recon 1. The model successfully predicted experimentally verified ATP and nitric oxide production rates in macrophages. This model was then integrated with an M. tuberculosis H37Rv model, iNJ661, to build an integrated host–pathogen genome-scale reconstruction, iAB-AMØ-1410-Mt-661. The integrated host–pathogen network enables simulation of the metabolic changes during infection. The resulting reaction activity and gene essentiality targets of the integrated model represent an altered infectious state. High-throughput data from infected macrophages were mapped onto the host–pathogen network and were able to describe three distinct pathological states. Integrated host–pathogen reconstructions thus form a foundation upon which understanding the biology and pathophysiology of infections can be developed.