Enzymes at the phosphoenolpyruvate (PEP)–pyruvate–oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and 13C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, Mycobacterium tuberculosis (Mtb). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO2 through the ANA node is essential for intracellular survival of Mtb and that Mtb possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of Mtb, which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.
High-throughput essentiality screens have enabled genome-wide assessments of the genetic requirements for growth and survival of a variety of bacteria in different experimental models. The reliance in many of these studies on transposon (Tn)-based gene inactivation has, however, limited the ability to probe essential gene function or design targeted screens. We interrogated the potential of targeted, large-scale, pooled CRISPR interference (CRISPRi)-based screens to extend conventional Tn approaches in mycobacteria through the capacity for positionally regulable gene repression. Here, we report the utility of the "CRISPRi-Seq" method for targeted, pooled essentiality screening, confirming strong overlap with TnSeq datasets. In addition, we exploit this high-throughput approach to provide insight into CRISPRi functionality. By interrogating polar effects and combining image-based phenotyping with CRISPRimediated depletion of selected essential genes, we demonstrate that CRISPRi-Seq can functionally validate Transcriptional Units within operons. Together, these observations suggest the utility of CRISPRiSeq to provide insights into (myco)bacterial gene regulation and expression on a genome-wide scale.
The causative agent of TB, Mycobacterium tuberculosis (Mtb) is once again the world’s number one infectious killer. M. tuberculosis resides primarily within macrophages and metabolic reprogramming within this intracellular niche is a crucial determinant of virulence. We previously applied the metabolic modelling-based tool 13C-flux spectral analysis (13C-FSA) to show that intracellular M. tuberculosis co-metabolises multiple gluconeogenic and glycolytic carbon substrates by utilizing the reactions of the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate (OAA) or anaplerotic (ANA) node. However, predicting the metabolic mode of operation required for intracellular survival is chellenging using a metabolic network as the ANA node consists of several apparently functionally redundant bidirectional reactions. Here we use multiple techniques including 13C isotopomer profiling, lipid analysis and fluorescent reporter strains to dissect the role of the ANA node. We show that this node has unexpected roles in the life cycle of M. tuberculosis including lipid biosynthesis, protection from known toxic intracellular carbon sources and redox regulation. Inhibiting enzymes at this node with novel therapeutic compounds restricts the growth of M. tuberculosis and limits the ability of this formidable pathogen to survive within the human host cell identifying the ANA node as a potential druggable pathway for controlling TB.
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