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

Julie Kovářová,Andrew W Pountain,Richard J S Burchmore,Stefan Weidt,David Wildridge,Michael P Barrett,Frédéric Bringaud,Fiona Achcar

doi:10.1371/journal.ppat.1006953

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.

Highlights

Leishmania are parasitic protozoa that cause a wide spectrum of diseases including a self-healing cutaneous form, a disfiguring mucocutaneous form and severe, often fatal, visceral leishmaniasis
Leishmania parasites endanger over 1 billion people worldwide, infecting 300,000 people and causing 20,000 deaths annually
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Summary

Introduction

Leishmania are parasitic protozoa that cause a wide spectrum of diseases including a self-healing cutaneous form, a disfiguring mucocutaneous form and severe, often fatal, visceral leishmaniasis. The individual clinical manifestation and severity of the disease depends on the causative Leishmania species, immuno-susceptibility of the patient and various additional factors [1]. Leishmania are transmitted by sandflies, and they alternate in their life-cycle between two major stages, the promastigote—insect stage—and amastigote—mammalian stage. The different stages vary significantly in their morphology and biochemistry, each of them adapted to their specific environment. Better understanding of the parasites’ biology is required with focus on new drug targets and treatment development. As disruption of essential biochemical pathways has instant detrimental effects on parasites, understanding metabolism offers a means to enhance our capability to intervene against the leishmaniases

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