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      Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana

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          Abstract

          Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not virulent in mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both.

          Author summary

          Leishmania parasites endanger over 1 billion people worldwide, infecting 300,000 people and causing 20,000 deaths annually. In this study, we scrutinized metabolism in Leishmania mexicana after deletion of the gene encoding transketolase (TKT), an enzyme involved in sugar metabolism via the pentose phosphate pathway which plays key roles in creating ribose 5-phosphate for nucleotide synthesis and also defence against oxidative stress. The insect stage of the parasite, grown in culture medium, did not suffer from any obvious growth defect after the gene was deleted. However, its metabolism changed dramatically, with metabolomics indicating profound changes to flux through the pentose phosphate pathway: decreased glucose consumption, and generally enhanced efficiency in using metabolic substrates with reduced secretion of partially oxidised end products of metabolism. This ‘stringent’ metabolism is reminiscent of the mammalian stage parasites. The cells were also more sensitive to oxidative stress inducing agents and leishmanicidal drugs. Crucially, mice inoculated with the TKT knock-out parasites did not develop an infection pointing to the enzyme playing a key role in allowing the parasites to remain viable in the host, indicating that TKT may be considered a useful target for development of new drugs against leishmaniasis.

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          The return of metabolism: biochemistry and physiology of the pentose phosphate pathway

          The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner–Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the ‘Warburg effect’ of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism.
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            Leishmaniasis: complexity at the host-pathogen interface.

            Leishmania is a genus of protozoan parasites that are transmitted by the bite of phlebotomine sandflies and give rise to a range of diseases (collectively known as leishmaniases) that affect over 150 million people worldwide. Cellular immune mechanisms have a major role in the control of infections with all Leishmania spp. However, as discussed in this Review, recent evidence suggests that each host-pathogen combination evokes different solutions to the problems of parasite establishment, survival and persistence. Understanding the extent of this diversity will be increasingly important in ensuring the development of broadly applicable vaccines, drugs and immunotherapeutic interventions.
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              The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro.

              Alamar Blue, an indicator for metabolic cell function, was evaluated as a fluorescent and as a colorimetric dye in drug sensitivity assays for human pathogenic African trypanosomes, Trypanosoma brucei rhodesiense and T.b. gambiense. The experimental conditions were adjusted to find those where the relationship between trypanosome number and Alamar Blue signal was linear over the widest possible range. Fluorescent signals correlated to trypanosome numbers from 10(4) trypanosomes/ml (T.b. rhodesiense) and 10(5) trypanosomes/ml (T.b. gambiense) up to 2-3 x 10(6) trypanosomes/ml when trypanosomes were incubated for 2 h with 10% Alamar Blue. Trypanocidal activity of common drugs (melarsoprol, DFMO, suramin, pentamidine and diminazene aceturate) was determined employing this assay. The IC50 values obtained were comparable to those obtained with another fluorochrome, BCECF-AM. The Alamar Blue assay can be applied for drug screening, since it is simple, reproducible and economical. The assay can also be used in field sites with less equipped laboratories, because in addition to fluorometric endpoint determination, a colorimetric reading is possible.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draft
                Role: InvestigationRole: Methodology
                Role: Data curationRole: Formal analysisRole: Methodology
                Role: ConceptualizationRole: Funding acquisitionRole: Supervision
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: Supervision
                Role: ConceptualizationRole: Data curationRole: Formal analysis
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                19 March 2018
                March 2018
                : 14
                : 3
                : e1006953
                Affiliations
                [1 ] Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
                [2 ] Glasgow Polyomics, Wolfson Wohl Cancer Research Centre, Garscube Campus, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
                [3 ] Centre de Résonance Magnétique des Systèmes Biologiques, Université de Bordeaux, Bordeaux, France
                Monash University, AUSTRALIA
                Author notes

                The authors have declared that no competing interests exist.

                [¤a]

                Current address: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom

                [¤b]

                Current address: Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia

                Author information
                http://orcid.org/0000-0001-8737-6403
                http://orcid.org/0000-0001-9651-5145
                http://orcid.org/0000-0003-1127-9214
                http://orcid.org/0000-0003-4552-6877
                http://orcid.org/0000-0001-8792-7615
                http://orcid.org/0000-0001-9447-3519
                Article
                PPATHOGENS-D-16-02805
                10.1371/journal.ppat.1006953
                5882173
                29554142
                1fcbfc5f-9961-41c7-85ee-ccbba6dead7d
                © 2018 Kovářová et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 16 December 2016
                : 28 February 2018
                Page count
                Figures: 12, Tables: 3, Pages: 31
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000780, European Commission;
                Award ID: 290080
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 104111/Z/14/Z
                Award Recipient :
                Funded by: Institutional Strategic Support Fund
                Award ID: 105614/Z/14/Z
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000275, Leverhulme Trust;
                Award Recipient :
                Funded by: Laboratoire d'Excellence
                Award ID: ANR-11-LABX-0024
                Award Recipient :
                This work was supported by Wellcome Trust with core grants to the Wellcome Centre for Molecular Parasitology (WCMP) 104111/Z/14/Z to MPB and the Institutional Strategic Support Fund (ISSF) 105614/Z/14/Z MPB, RJSB and SW. JK was funded by the European Commission FP7 Marie Curie Initial Training Network “ParaMet” [grant number 290080]. FA is funded by a Leverhulme Trust early career fellowship. FB was supported by the Laboratoire d’Excellence (LabEx) ParaFrap (grant number ANR-11-LABX-0024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Carbohydrate Metabolism
                Glucose Metabolism
                Biology and Life Sciences
                Developmental Biology
                Life Cycles
                Protozoan Life Cycles
                Promastigotes
                Biology and Life Sciences
                Microbiology
                Protozoology
                Protozoan Life Cycles
                Promastigotes
                Biology and Life Sciences
                Cell Biology
                Cell Physiology
                Cell Metabolism
                Medicine and Health Sciences
                Parasitic Diseases
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Carbohydrates
                Monosaccharides
                Glucose
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Carbohydrates
                Monosaccharides
                Glucose
                Biology and Life Sciences
                Biochemistry
                Metabolism
                Metabolomics
                Medicine and Health Sciences
                Pharmacology
                Pharmacokinetics
                Drug Metabolism
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzyme Chemistry
                Enzyme Metabolism
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-04-03
                Metabolomics data have been deposited to the EMBL-EBI MetaboLights database (DOI: 10.1093/nar/gks1004. PubMed PMID: 23109552) with the identifier MTBLS491.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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