A systems chemical biology study of malate synthase and isocitrate lyase inhibition in Mycobacterium tuberculosis during active and NRP growth

The ability of Mycobacterium tuberculosis (Mtb) to survive in low oxygen environments enables the bacterium to persist in a latent state within host tissues. In vitro studies of Mtb growth have identified changes in isocitrate lyase (ICL) and malate synthase (MS) that enable bacterial persistent under low oxygen and other environmentally limiting conditions. Systems chemical biology (SCB) enables us to evaluate the effects of small molecule inhibitors not only on the reaction catalyzed by malate synthase and isocitrate lyase, but the effect on the complete tricarboxylic acid cycle (TCA) by taking into account complex network relationships within that system

Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic Mycobacterium tuberculosis

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis

Glyoxylate Metabolism in Mycobacterium smegmatis

Much has been learned about Mycobacterium tuberculosis, the causative agent of tuberculosis, “the great white plague,†since the bacterium was isolated and initially characterized by Robert Koch over a century ago. Over the last decade, new genetic tools for manipulation of the bacterium have been developed, its genome has been sequenced, and the search for new vaccines and drug targets has greatly intensified. Yet, surprisingly little is known about which mycobacterial genes are truly important for the organism’s ability to persist in the tissues of its human hosts. The metabolic pathways used by the tubercle bacillus to establish and maintain a life-long infection have largely been ignored by researchers, yet they may represent promising new areas for therapeutic intervention. Recently, one enzyme of the glyoxylate shunt of M. tuberculosis, isocitrate lyase (ICL), was shown to be required for virulence in experimental infections of mice. The other enzyme of the glyoxylate shunt, malate synthase (MLS), may also be important for the intracellular survival of the tubercle bacillus; yet, no studies have been done to determine its in vivo role. We present here results of genetic studies of MLS in the saprophyte Mycobacterium smegmatis, and show that MLS, unlike ICL, is dispensable for growth on acetate or fatty acids. We also describe the dglycerate pathway in M. smegmatis, which enables malate synthase-deficient bacteria to utilize acetate and fatty acids as sole carbon sources, and which allows M. smegmatis to grow on glyoxylate. The d-glycerate pathway, however, does not appear to exist in the pathogenic Mycobacterium tuberculosis

Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis</it>, the causative agent of tuberculosis (TB), infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant <it>M. tuberculosis </it>strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections.</p> <p>Methods</p> <p>The availability of <it>M. tuberculosis </it>genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality.</p> <p>Results</p> <p>Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by <it>devR</it>, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (<it>devR/devS</it>, <it>relA</it>, <it>mprAB</it>), enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability, and drug development.</p> <p>Conclusion</p> <p>Based on our bioinformatics analysis and additional discussion of in-depth biological rationale, several novel anti-TB targets have been proposed as potential opportunities to improve present therapeutic treatments for this disease.</p

Regulation of the Branch point between the glyoxylate shunt and the TCA cycle in mycobacteria

Master'sJOINT M.SC. IN INFECTIOUS DISEASES, VACCINOLOGY AND DRUG DISCOVER

Molecular, biochemical and pharmacological characterisation of Mycobacterium tuberculosis cytochrome bd-I oxidase: a putative therapeutic target

Tuberculosis (TB) remains one of the most devastating diseases in humans. Nowadays, tuberculosis therapy is not sufficient to control the TB epidemic and only lasts for 6 months to cure patients and prevent relapse; therefore, the treatment of Mycobacterium tuberculosis (Mtb) is particularly challenging (1). New antibiotics, mainly those that are derived from new chemical classes, are more likely to be more effective against resistant strains. Moreover, expanding the knowledge of the mode of action of drugs has important implications in tackling TB. Only empirical approaches can be adopted in the journey of discovering new anti-tubercular drugs until a clear picture of latency and persister cells’ physiology is achieved. Mtb has the extraordinary ability to survive under hypoxia, suggesting a high degree of metabolic plasticity. The flexibility conferred by a modular respiratory system is critical to the survival of Mtb, thereby also making it a promising area of research for new drug targets. This thesis aimed towards the characterisation of cytochrome bd-I quinol oxidase (bd-I), a respiratory component that is believed to operate during both the replicative and “dormant” Mtb phenotypes. The essential nature of Mtb bd-I, which has no human homologue, has been confirmed in a recent deep sequencing study of genes required for Mtb growth by Griffin et al. (2), further confirming its potential as a novel target. Recombinant Mtb bd-I was successfully expressed under the control of the pUC19 lac promoter in the Escherichia coli ML16 bo3/bd-I and MB44 bo3/bd-I/bd-II knockout strains, allowing “noise-free” measurement of the enzyme. Initial steady-state kinetics of the enzyme was presented using a range of quinol substrates, revealing a substrate preference for dQH2 over Q1H2 and Q2H2. A number of bd-I inhibitors were identified and their pharmacodynamic profiles against Mtb H37Rv were determined. In addition, a pharmaco-metabolomics platform was initiated to explore the cellular response of Mtb to current first-line TB drugs as well as in house bd-I and type II NADH inhibitors. The initial findings are discussed in the context of the known mode of action of the drugs and future research needs in drug discovery of this devastating disease

Functional and mathematical analysis of the glyoxylate shunt in Streptomyces coelicolor

Streptomyces coelicolor is the model organism for the genus Streptomyces, which produces many bioactive secondary metabolites with clinical applications. Based on work done in Escherichia coli, the glyoxylate shunt was thought to be the main anapleurotic pathway in S. coelicolor during growth on fatty acids and therefore an important pathway in providing precursors for secondary metabolism. The S. coelicolor genome contains genes for a second anapleurotic pathway, the ethylmalonyl-CoA pathway. The relative importance of both to anapleurosis in streptomycete metabolism was unclear. The function of the glyoxylate shunt was investigated in this thesis using sequence analysis, genetic manipulation, transcriptomics and mathematical modelling. Analysis of orthologues of aceA, ccr and genes encoding tricarboxylic acid (TCA) cycle genes revealed that all are subject to a similar level of purifying selection pressure. The operons of the glyoxylate shunt and the ethylmalonyl-CoA pathway share a 15 bp palindromic motif in their upstream sequences, which was also found upstream of other genes. This suggests an overlap in regulation and thus an overlap in function. The sequence analysis is contradicted by results of experiments with an aceA⁻ aceB1⁻ mutant, which did not display a phenotype during growth on Tween 40, a model carbon source for fatty acids. Results obtained by total RNA sequencing indicate that the ethylmalonyl-CoA pathway is the main anapleurotic pathway during growth of S. coelicolor on fatty acids whereas expression of the glyoxylate shunt is minimal. This apparent contradiction is resolved by hypothesising that the ethylmalonyl-CoA pathway is the main anapleurotic pathway, but that the glyoxylate shunt provides a backup when acyl-CoA thioesters are withdrawn from the ethylmalonyl-CoA pathway for secondary metabolite biosynthesis. Enzymes of the isocitrate branchpoint were isolated following heterologous expression and analysed. The resulting kinetic parameters, as well as their specific activities measured during growth on Tween 40 and additional data from literature, were used to set up a mathematical model of the TCA cycle and the glyoxylate shunt. Simulations of this model predicted that, as growth proceeds from early to mid and late exponential phase, the relative concentrations of TCA cycle intermediates changed from promoting gluconeogenesis to accomodating secondary metabolism. Further model refinement is needed using data on the flux through the ethylmalonyl- CoA pathway as these were unavailable at the time of writing.Streptomyces coelicolor is the model organism for the genus Streptomyces, which produces many bioactive secondary metabolites with clinical applications. Based on work done in Escherichia coli, the glyoxylate shunt was thought to be the main anapleurotic pathway in S. coelicolor during growth on fatty acids and therefore an important pathway in providing precursors for secondary metabolism. The S. coelicolor genome contains genes for a second anapleurotic pathway, the ethylmalonyl-CoA pathway. The relative importance of both to anapleurosis in streptomycete metabolism was unclear. The function of the glyoxylate shunt was investigated in this thesis using sequence analysis, genetic manipulation, transcriptomics and mathematical modelling. Analysis of orthologues of aceA, ccr and genes encoding tricarboxylic acid (TCA) cycle genes revealed that all are subject to a similar level of purifying selection pressure. The operons of the glyoxylate shunt and the ethylmalonyl-CoA pathway share a 15 bp palindromic motif in their upstream sequences, which was also found upstream of other genes. This suggests an overlap in regulation and thus an overlap in function. The sequence analysis is contradicted by results of experiments with an aceA⁻ aceB1⁻ mutant, which did not display a phenotype during growth on Tween 40, a model carbon source for fatty acids. Results obtained by total RNA sequencing indicate that the ethylmalonyl-CoA pathway is the main anapleurotic pathway during growth of S. coelicolor on fatty acids whereas expression of the glyoxylate shunt is minimal. This apparent contradiction is resolved by hypothesising that the ethylmalonyl-CoA pathway is the main anapleurotic pathway, but that the glyoxylate shunt provides a backup when acyl-CoA thioesters are withdrawn from the ethylmalonyl-CoA pathway for secondary metabolite biosynthesis. Enzymes of the isocitrate branchpoint were isolated following heterologous expression and analysed. The resulting kinetic parameters, as well as their specific activities measured during growth on Tween 40 and additional data from literature, were used to set up a mathematical model of the TCA cycle and the glyoxylate shunt. Simulations of this model predicted that, as growth proceeds from early to mid and late exponential phase, the relative concentrations of TCA cycle intermediates changed from promoting gluconeogenesis to accomodating secondary metabolism. Further model refinement is needed using data on the flux through the ethylmalonyl- CoA pathway as these were unavailable at the time of writing

The effect of self-generated hypoxia on the expression of target genes coding for electron transport related products in mycobacterium tuberculosis.

Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2010.The work presented here aims at identifying whether the genes identified in the genome of Mycobacterium tuberculosis that code for products involved in anaerobic metabolism are active or inactivated genes. The study consists of three distinct parts. In part one, serial dilutions of sputum of patients with pulmonary tuberculosis (PTB) were grown on agar surface and in high columns of un-agitated broth. The highest dilution from which mycobacteria was grown was for all patients significantly higher in the broth cultures than on the plates suggesting the presence of anaerobically metabolizing mycobacteria in the lungs of patients with PTB. Part two of the study identified gene expression by measuring the concentration of transcripts for 5 genes involved in aerobic or anaerobic pathways. This was done over a period of 15 weeks using un-agitated broth cultures (the Wayne method). Undulating patterns of gene expression were found with the genes coding for anaerobic metabolic pathway components expressed at higher levels than those coding for aerobic pathway components while the cultures grew older. Part three aimed at measuring transcription products of the same set of genes directly in sputum specimens. Although quantitation at bacterial cell level in the sputum could not be achieved, expression of all genes was established with on average larger quantities of transcripts of genes coding for the anaerobic pathway components

Genetic Identification of Novel Mycobacterium tuberculosis Susceptibility and Survival Mechanisms During Antibiotic Treatment

Effective treatment of tuberculosis requires at least six months of combination therapy involving four antibiotics. Alterations in the physiological state of Mycobacterium tuberculosis during infection may reduce drug efficacy and prolong treatment, but these adaptations are incompletely defined. To investigate the mechanisms limiting antibiotic efficacy, I performed a comprehensive genetic study to identify M. tuberculosis genes and pathways important for bacterial survival during antibiotic treatment in vivo. First, I identified mutants in the glycerol kinase enzyme, GlpK, that promote survival under combination therapy. Similar glycerol catabolic mutants are enriched in extensively drug-resistant clinical isolates, indicating that these mutations may promote survival and the development of resistance in humans. A majority of these mutations are frameshifts within a homopolymeric region of the glpK gene, leading to the hypothesis that M. tuberculosis may reversibly produce drug-tolerant phenotypes through genetic variation introduced at homopolymer sites as a strategy for survival during antibiotic treatment. Second, I identified bacterial mutants with altered susceptibility to individual first-line anti-mycobacterial drugs. Many of these mutations did not have obvious effects in vitro, demonstrating that a wide variety of natural genetic variants can influence drug efficacy in vivo without altering standard drug-susceptibility tests. A number of these genes are enriched in drug-resistant clinical isolates, indicating that these genetic variants influence treatment outcome. Together, these data suggest new targets for improving therapy, as well as mechanisms of genetic adaptations that can reduce antibiotic efficacy and contribute to the evolution of resistance