Machine learning and structural analysis of Mycobacterium tuberculosis pan-genome identifies genetic signatures of antibiotic resistance.

Mycobacterium tuberculosis is a serious human pathogen threat exhibiting complex evolution of antimicrobial resistance (AMR). Accordingly, the many publicly available datasets describing its AMR characteristics demand disparate data-type analyses. Here, we develop a reference strain-agnostic computational platform that uses machine learning approaches, complemented by both genetic interaction analysis and 3D structural mutation-mapping, to identify signatures of AMR evolution to 13 antibiotics. This platform is applied to 1595 sequenced strains to yield four key results. First, a pan-genome analysis shows that M. tuberculosis is highly conserved with sequenced variation concentrated in PE/PPE/PGRS genes. Second, the platform corroborates 33 genes known to confer resistance and identifies 24 new genetic signatures of AMR. Third, 97 epistatic interactions across 10 resistance classes are revealed. Fourth, detailed structural analysis of these genes yields mechanistic bases for their selection. The platform can be used to study other human pathogens

Structure-guided Inhibitor Design of Mycobacterium Tuberculosis Drug Targets from Central Carbon Metabolism

A key determinant of the pathogenicity of M. tuberculosis (Mtb) is its ability to conserve energy and resources by altering its intermediate metabolism response to host-derived hostile conditions. Growing evidence supports that the central carbon metabolism (CCM) of M.tuberculosis including glycolysis, gluconeogenesis, the pentose phosphate pathway, and the TCA cycle, is regulated to allow for simultaneous utilization of carbohydrates and fatty acids-derived carbon sources. The genes involved in CCM represent attractive anti TB drug-targets. Although the role of Mtb phosphoenolpyruvate carboxykinase (PEPCK) in gluconeogenesis pathway and pyruvate kinase (PykA) enzymes in glycolysis pathway are relevant for the M. tuberculosis pathogenesis, targeting these enzymes for the drug development is not straight forward because of the human orthologs existence. Here, X-ray crystallographic, biochemical, and inhibitory studies of Mtb PEPCK and PykA highlight distinct features of the pathogenic’s enzyme that differ from those of the host orthologs’ and provide opportunities to develop selective and potent inhibitors. Structural data of Mtb PEPCK has revealed that the conformation changes of the flexible loops in response to substrates binding differ from the one reported for host versions of the enzyme. GTP-competitive inhibitors of human cytosolic PEPCK bind to Mtb PEPCK in similar fashion, but the discovery of two unique small pockets in Mtb has created an opportunity for the design of a selective Mtb PEPCK inhibitor. By using structure-guided medicinal chemistry for GTP-competitive inhibitor series with PEPCK, we are able to improve the inhibitory effect of these inhibitors against the enzyme, creating inhibitors with a nano-molar range IC50 and develop 10 fold selective inhibitors against Mtb PEPCK over human PEPCK. Structural study of Mtb PykA also demonstrated that adenosine mono-phosphate (AMP) is an allosteric effector, and we identified the binding mode and interaction of AMP in the allosteric binding site for the first time. Screening diverse compound libraries gave us insight into the potential scaffolds for the development of more potent pathogen-specific inhibitors. The structural and inhibitor studies of Mtb PEPCK and PykA have allowed a better understanding of their role in gluconeogenesis and glycolysis, and provide a framework for the development of selective inhibitors

Modeling metabolism of Mycobacterium tuberculosis

Approximately one-fourth of the Mycobacterium tuberculosis (Mtb) genome contains genes that encode enzymes directly involved in its metabolism. These enzymes represent potential drug targets that can be systematically probed with constraint based (CB) models through the prediction of genes essential (or the combination thereof) for the pathogen to grow. However, gene essentiality depends on the growth conditions and, so far, no in vitro model precisely mimics the host at the different stages of mycobacterial infection, limiting model predictions. A first step in creating such a model is a thoroughly curated and extended genome-scale CB metabolic model of Mtb metabolism. The history of genome-scale CB models of Mtb metabolism up to model sMtb are discussed and sMtb is quantitatively validated using 13C measurements. The human pathogen Mtb has the capacity to escape eradication by professional phagocytes. During infection, Mtb resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to many other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio. The human macrophage-like cell line THP-1 was infected with the attenuated Mtb surrogate Mycobacterium bovis Bacillus Calmette–Guérin (M. bovis BCG). Mycobacterial RNA was up to 1000-fold underrepresented in total RNA preparations of infected host cells. By combining microbial enrichment with specific ribosomal RNA depletion the transcriptional responses of host and pathogen during infection were simultaneously analyzed using dual RNA sequencing. Mycobacterial pathways for cholesterol degradation and iron acquisition are upregulated during infection. In addition, genes involved in the methylcitrate cycle, aspartate metabolism and recycling of mycolic acids are induced. In response to M. bovis BCG infection, host cells upregulate de novo cholesterol biosynthesis presumably to compensate for the loss of this metabolite by bacterial catabolism. By systematically probing the metabolic network underpinning sMtb, the reactions that are essential for Mtb are identified. A majority of these reactions are catalyzed by enzymes and thus represent candidate drug targets to fight an Mtb infection. Modeling the behavior of the bacteria during infection requires knowledge of the so-called biomass reaction that represents bacterial biomass composition. This composition varies in different environments or bacterial growth phases. Accurate modeling of all fluxes through metabolism under a given condition at a moment in time, the so called metabolic state, requires a precise description of the biomass reaction for the described condition. The transcript abundance data obtained by dual RNA sequencing was used to develop a straightforward and systematic method to obtain a condition-specific biomass reaction for Mtb during in vitro growth and during infection of its host. The method described herein is virtually free of any pre-set assumptions on uptake rates of nutrients, making it suitable for exploring environments with limited accessibility. The condition-specific biomass reaction represents the 'metabolic objective' of Mtb in a given environment (in-host growth and growth on defined medium) at a specific time point, and as such allows modeling the bacterial metabolic state in these environments. Five different biomass reactions were used predict nutrient uptake rates and gene essentiality. Predictions were subsequently compared to available experimental data. Nutrient uptake can accurately be predicted, but accurate gene essentiality predictions remain difficult to obtain. By combining sMtb and a model of human metabolism, model sMtb-RECON was developed and used to predict the metabolic state of Mtb during infection of the host. Amino acids are predicted to be used for energy production as well as biomass formation. Subsequently the effect of increasing dosages of drugs, targeting metabolism, on the metabolic state of the pathogen was assessed and resulting metabolic adaptations and flux rerouting through various pathways is predicted. In particular, the TCA cycle becomes more important upon drug application, as well as alanine, aspartate, glutamate, proline, arginine and porphyrin metabolism, while glycine, serine and threonine metabolism become less important for survival. Notably, an effect of eight out of eleven metabolically active drugs could be recreated and two major profiles of the metabolic state were predicted. The profiles of the metabolic states of Mtb affected by the drugs BTZ043, cycloserine and its derivative terizidone, ethambutol, ethionamide, propionamide, and isoniazid were very similar, while TMC207 is predicted to have quite a different effect on metabolism as it inhibits ATP synthase and therefore indirectly interferes with a multitude of metabolic pathways.</p

Visualising the subcellular distribution of antibiotics against tuberculosis

Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), remains the world’s deadliest infectious disease. Although treatable, effective chemotherapy requires at least six months of treatment with a minimum of four antibiotics. Novel antibiotics are needed to quell the pandemic. However, we do not fully understand why current treatments take so long to work in patients. Mtb has a dynamic intracellular lifestyle, and this thesis tests the hypothesis that not all antibiotics penetrate into, or are effective within, all compartments containing Mtb during infection. Our understanding of the intracellular pharmacokinetics of drugs against TB has been limited by a lack of technologies for studying the subcellular distribution of antibiotics. This work developed a correlative imaging workflow incorporating fluorescence, electron and nanoscale ion microscopy (CLEIM) to map the subcellular distribution of two antibiotics, bedaquiline (BDQ) and pyrazinamide (PZA), at sub-micrometre resolution in Mtb-infected human macrophages. This workflow was complemented with orthogonal methods, including high-content live-cell imaging, to study the dynamic processes that contribute to antibiotic activity. BDQ accumulated primarily in host cell lipid droplets (LD), but heterogeneously in Mtb within a variety of intracellular compartments. Surprisingly, LD did not sequester the antibiotic but constituted a transferable reservoir that enhanced antibacterial efficacy. Lipid binding therefore facilitated drug trafficking by host organelles to an intracellular target. PZA is a pro-drug, and the accumulation of its active metabolite pyrazinoic acid has been hypothesised to depend on the bacteria being in an acidic environment. Direct analysis of antibiotic accumulation by ion microscopy, combined with live-cell imaging at the single cell level, revealed that, whilst acidic intracellular environments support PZA activity, they are not necessary for antibiotic efficacy. Many intracellular pathogens interact with LD or reside in partially acidified vacuoles, and these results therefore have broad implications for our understanding of antibiotic activity

The Role of the Glyoxylate Cycle in the Pathogenesis of Mycobacterium tuberculosis

According to the World Health Organization a third of the world\u27s population is infected with Mycobacterium tuberculosis. The unparalleled success of M. tuberculosis as a pathogen reflects the bacterium\u27s extraordinary ability to persist in its host in spite of eliciting a robust immune response. Currently available treatment is inadequate and drug resistance is rapidly spreading. New antibiotics are desperately needed. The substrates and metabolic pathways utilized by pathogens during infection are largely unknown and represent an under-exploited area of investigation. Uniquely, evolution of the genus Mycobacterium has involved extensive duplication of fatty acid metabolism genes, including two homologs encoding prokaryotic- and eukaryotic-like isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL). The glyoxylate cycle is employed by cells when fatty acids are the main carbon source available. Here, we show that these enzymes are jointly required by M. tuberculosis for growth on fatty acids and for virulence in experimental infections. Although deletion of icll or icl2 had little impact on replication of M. tuberculosis in macrophages and mice, deletion of both genes abrogated intracellular growth, and resulted in rapid bacterial clearance from the lungs. A dual-specificity ICL inhibitor similarly blocked replication of M. tuberculosis on fatty acids in vitro and in macrophages. The absence of ICL orthologs in mammals, and recent findings implicating the glyoxylate pathway in the virulence of other bacterial and fungal pathogens makes this metabolic pathway an attractive novel target for drug development

Molecular and biochemical characterisation of key enzymes involved in mycolic acid biosynthesis from Mycobacterium tuberculosis

PhD ThesisMycolic acids are the dominant feature of the Mycobacterium tuberculosis cell wall, providing the basis for its lipid-rich permeability barrier. These oc-alkyl, P-hydroxy fatty acids are thought to be formed by the Claisen-typec ondensationo f a long C56m eromycolic acid and a shorter C24-C2f6a tty acid. These componentf atty acids are producedv ia a combination of type I and 11f atty acid synthase( FAS) systems.T he C16-C2f6a tty acyl products of FAS-I are elongated by FAS-II with simultaneous modification to form meromycolic acids, which are then condensedw ith the C24-C26fa tty acyl chain. These studies aimed to characterisek ey enzymes of FAS-11 (mtFabH, KasA) and enzymes possibly involved in the Claisen-type condensationr eactiont o form mycolatic acids (Accl) enzymes,P ksl3, FadD32). The P-ketoacyl ACP synthase (KAS) III (mtFabH) is proposed to link FAS-I and FAS-11, catalyzing the condensation of FAS-1-derived acyl-CoA with malonyl-Acyl Carrier Protein (ACP). The acyl-CoA chain length specificity of mtFabH was assessedin vitro . When using E. coll, the preferreds ubstratesw ere C12-a ndC 14-CoAH. owever, with the mycobacterialA CP (AcpM), the enzyme was able to utilise longer (up tp C2o) acyl-CoA chains. The substitution of residues implicated in acyl-CoA chain length specificity totally abrogated overall KAS activity and reduced the transacylation activity of the enzyme. Mutation of the proposed catalytic triad residues confirmed that Cys122 is essential for transacylation and His258 is essential for malonyl-AcpM decarboxylation. KasA, which belongs to the FAS-11 system, utilises palmitoyl-ACP rather than short-chain acyl-ACP primers. Purified recombinant KasA had in vitro KAS activity that was highly sensitive to cerulenin, a well-known KAS inhibitor. Mutation of proposed catalytic residues Cys 17 1, His31 1, Lys340 and His345 inactivated the enzyme completely. Four putative accD genes were found in Corynebacterium glutamicum. Overexpression of each gene resulted in increased acyl-CoA dependent 14CO2 fixation in vitro, providing evidence that the accD genes encode a family of carboxyltransferases. Disruption of either accD2 or accD3 led to complete and specific loss of mycolic acids. These two carboxyltransferasesa re also retained in all Corynebacterianeaei,n cluding M. leprae, and probably provide a carboxylated. intennediate for condensation of the mero-chain and CCbranch directed by thepks]3-encoded polyketide synthase

Identification of a Novel Mycobacterial Gene Involved in the Synthesis of a Phenolic Glycolipid and its Role in the Prevention of Phagosome Maturation

Pathogenic Mycobacteria persist in an early endosome-like compartment by interfering with late endosomal fusion mediating factors. Studies have unraveled some of the mechanisms employed by mycobacteria to create a niche for themselves in macrophages, but it is widely accepted that they possess an arsenal of weapons to impede phagosome genesis. M. marinum has gained importance in recent years, as a model organism to study mycobacterial pathogenesis due to its phylogenetic closeness to M. tuberculosis. The infection it causes in its natural hosts display characteristic features of tuberculosis, exhibiting blocking of phagosome maturation and granuloma formation. To gain insight into the genes required for the inhibition of phagosome maturation, M. marinum transposon mutant library representing knock outs covering the entire genome was sifted for mutants defective in inhibiting phagosome maturation by designing an elegant screen, which employs magnetic separation. In this process we identified a number of mutants unable to inhibit phagosome maturation and characterised in detail one of these mutants (mutant P1). The colony morphology and sequence analysis revealed that the interrupted gene of mutant P1 (pmiA) is likely to be involved in lipid metabolism. The mutant also had a reduced intracellular survival as inferred from the in vitro bacterial survival experiments in HMDM and using mice as an in vivo model. The mutant completely reverted to its wild-type phenotype when complemented with the respective gene from wild-type M. marinum. Thin layer chromatography on the lipids isolated from the mutant showed that the disruption of the gene pmiA in mutant P1 leads to the loss of a glycolipid of the outer envelope of M. marinum (Robinson N et al., Infect Immun. 2007 Feb;75(2):581-91). The missing glycolipid was further characterised to be a phenolic glycolicpid (PGL) using mass spectrometry and nuclear magnetic resonance spectroscopy. In order to prove that the lipid is capable of inhibiting phagosome maturation, it was extracted from wild-type M. marinum, coated on to hydrophobic beads and chased into human monocyte derived macrophages (HMDM). Characterising the phagosomes containing the beads by western blot analysis and immunofluorescence microscopy proved the lipid to be a key molecule employed by virulent mycobacteria to inhibit phagosome maturation. Phagosomes were characterised employing an efficient adenoviral transfection system harbouring Rab-GFP fusion proteins to transfect primary phagocytes. This transfection system enables phagosome maturation to be studied efficiently by fluorescence microscopy in live cells, in contrast to immunostaining which can be performed only on fixed cells. The gene pmiA involved in the biosynthesis of the phenolic glycolipid shows little homology with the gene sequences available through genome databases. It also does not display any signature sequences of proteins with known functions. Therefore, an attempt was made to study its interacting proteins by using Histidine-tag pull down assay. Proteins interacting with pmiA were analyzed by mass spectrometry. A methyl transferase and an isocitrate lyase, both enzymes critically involved in lipid biosynthesis were found to interact with pmiA. Our results prove that genes involved in the synthesis of this phenolic glycolipid are ideal pharmacological targets to design drug interventions against tuberculosis