Reconciling high-throughput gene essentiality data with metabolic network reconstructions.

The identification of genes essential for bacterial growth and survival represents a promising strategy for the discovery of antimicrobial targets. Essential genes can be identified on a genome-scale using transposon mutagenesis approaches; however, variability between screens and challenges with interpretation of essentiality data hinder the identification of both condition-independent and condition-dependent essential genes. To illustrate the scope of these challenges, we perform a large-scale comparison of multiple published Pseudomonas aeruginosa gene essentiality datasets, revealing substantial differences between the screens. We then contextualize essentiality using genome-scale metabolic network reconstructions and demonstrate the utility of this approach in providing functional explanations for essentiality and reconciling differences between screens. Genome-scale metabolic network reconstructions also enable a high-throughput, quantitative analysis to assess the impact of media conditions on the identification of condition-independent essential genes. Our computational model-driven analysis provides mechanistic insight into essentiality and contributes novel insights for design of future gene essentiality screens and the identification of core metabolic processes.

Glucosylceramide synthase modulation ameliorates murine renal pathologies and promotes macrophage effector function in vitro.

While significant advances have been made in understanding renal pathophysiology, less is known about the role of glycosphingolipid (GSL) metabolism in driving organ dysfunction. Here, we used a small molecule inhibitor of glucosylceramide synthase to modulate GSL levels in three mouse models of distinct renal pathologies: Alport syndrome (Col4a3 KO), polycystic kidney disease (Nek8jck), and steroid-resistant nephrotic syndrome (Nphs2 cKO). At the tissue level, we identified a core immune-enriched transcriptional signature that was shared across models and enriched in human polycystic kidney disease. Single nuclei analysis identified robust transcriptional changes across multiple kidney cell types, including epithelial and immune lineages. To further explore the role of GSL modulation in macrophage biology, we performed in vitro studies with homeostatic and inflammatory bone marrow-derived macrophages. Cumulatively, this study provides a comprehensive overview of renal dysfunction and the effect of GSL modulation on kidney-derived cells in the setting of renal dysfunction.

Reconstruction of the metabolic network of Pseudomonas aeruginosa to interrogate virulence factor synthesis.

Virulence-linked pathways in opportunistic pathogens are putative therapeutic targets that may be associated with less potential for resistance than targets in growth-essential pathways. However, efficacy of virulence-linked targets may be affected by the contribution of virulence-related genes to metabolism. We evaluate the complex interrelationships between growth and virulence-linked pathways using a genome-scale metabolic network reconstruction of Pseudomonas aeruginosa strain PA14 and an updated, expanded reconstruction of P. aeruginosa strain PAO1. The PA14 reconstruction accounts for the activity of 112 virulence-linked genes and virulence factor synthesis pathways that produce 17 unique compounds. We integrate eight published genome-scale mutant screens to validate gene essentiality predictions in rich media, contextualize intra-screen discrepancies and evaluate virulence-linked gene distribution across essentiality datasets. Computational screening further elucidates interconnectivity between inhibition of virulence factor synthesis and growth. Successful validation of selected gene perturbations using PA14 transposon mutants demonstrates the utility of model-driven screening of therapeutic targets.

Integration of expression data in genome-scale metabolic network reconstructions.

With the advent of high-throughput technologies, the field of systems biology has amassed an abundance of "omics" data, quantifying thousands of cellular components across a variety of scales, ranging from mRNA transcript levels to metabolite quantities. Methods are needed to not only integrate this omics data but to also use this data to heighten the predictive capabilities of computational models. Several recent studies have successfully demonstrated how flux balance analysis (FBA), a constraint-based modeling approach, can be used to integrate transcriptomic data into genome-scale metabolic network reconstructions to generate predictive computational models. In this review, we summarize such FBA-based methods for integrating expression data into genome-scale metabolic network reconstructions, highlighting their advantages as well as their limitations.

Metabolic network analysis predicts efficacy of FDA-approved drugs targeting the causative agent of a neglected tropical disease.

BACKGROUND: Systems biology holds promise as a new approach to drug target identification and drug discovery against neglected tropical diseases. Genome-scale metabolic reconstructions, assembled from annotated genomes and a vast array of bioinformatics/biochemical resources, provide a framework for the interrogation of human pathogens and serve as a platform for generation of future experimental hypotheses. In this article, with the application of selection criteria for both Leishmania major targets (e.g. in silico gene lethality) and drugs (e.g. toxicity), a method (MetDP) to rationally focus on a subset of low-toxic Food and Drug Administration (FDA)-approved drugs is introduced. RESULTS: This metabolic network-driven approach identified 15 L. major genes as high-priority targets, 8 high-priority synthetic lethal targets, and 254 FDA-approved drugs. Results were compared to previous literature findings and existing high-throughput screens. Halofantrine, an antimalarial agent that was prioritized using MetDP, showed noticeable antileishmanial activity when experimentally evaluated in vitro against L. major promastigotes. Furthermore, synthetic lethality predictions also aided in the prediction of superadditive drug combinations. For proof-of-concept, double-drug combinations were evaluated in vitro against L. major and four combinations involving the drug disulfiram that showed superadditivity are presented. CONCLUSIONS: A direct metabolic network-driven method that incorporates single gene essentiality and synthetic lethality predictions is proposed that generates a set of high-priority L. major targets, which are in turn associated with a select number of FDA-approved drugs that are candidate antileishmanials. Additionally, selection of high-priority double-drug combinations might provide for an attractive and alternative avenue for drug discovery against leishmaniasis.