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

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.

Leishmania Encodes a Bacterium-like 2,4-Dienoyl-Coenzyme A Reductase That Is Required for Fatty Acid β-Oxidation and Intracellular Parasite Survival

“…7C). However, as often observed with attenuated Leishmania mutant lines (36)(37)(38), limiting dilution culturing from tissues at the site of injection revealed that some L. major Δdecr parasites persisted 8 weeks post in vivo infection. Thus, DECR is a virulence factor and decr-deficient parasites were unable to cause lesions in a highly susceptible host.…”

Mining for natural product antileishmanials in a fungal extract library

“…For LC-MS metabolomic analyses, sample extraction was performed as described (Creek et al, 2011; Kovářová et al, 2018). In summary, 10 8 cells were rapidly cooled in a dry ice/ethanol bath to 4 °C, centrifuged, washed with 1 × PBS, and resuspended in 200 μL extraction solvent (chloroform:methanol:water, 1:3:1 vol ratio).…”

Transcriptional shift and metabolic adaptations during Leishmania quiescence using stationary-phase and drug pressure as models

“…While the abundance of carbon sources such as sucrose and 3 hexose-multimers (labelled as cellopentaose, cellohexaose and celloheptaose) were identified as increasing in quiescent cells. These penta, hexa and heptahexoses have previously been inferred [31] to derive from mannogen, a poly-mannose carbohydrate reserve used by Leishmania [32]. Interestingly, while most structural phospholipids were not modulated, a number of free fatty acyls and also sphingolipids were increased in quiescent promastigotes, while no clear trend was observed in amastigotes.…”