Biochemistry and Pharmacology - Research Publications
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ItemLeishmania mexicana can utilize amino acids as major carbon sources in macrophages but not in animal models(WILEY, 2018-04)Leishmania parasites target macrophages in their mammalian hosts and proliferate within the mature phagolysosome compartment of these cells. Intracellular amastigote stages are dependent on sugars as a major carbon source in vivo, but retain the capacity to utilize other carbon sources. To investigate whether amastigotes can switch to using other carbon sources, we have screened for suppressor strains of the L. mexicana Δlmxgt1-3 mutant which lacks the major glucose transporters LmxGT1-3. We identified a novel suppressor line (Δlmxgt1-3s2 ) that has restored growth in rich culture medium and virulence in ex vivo infected macrophages, but failed to induce lesions in mice. Δlmxgt1-3s2 amastigotes had lower rates of glucose utilization than the parental line and primarily catabolized non-essential amino acids. The increased mitochondrial metabolism of this line was associated with elevated levels of intracellular reactive oxygen species, as well as increased sensitivity to inhibitors of the tricarboxylic acid (TCA) cycle, including nitric oxide. These results suggest that hardwired sugar addiction of Leishmania amastigotes contributes to the intrinsic resistance of this stage to macrophage microbicidal processes in vivo, and that these stages have limited capacity to switch to using other carbon sources.
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ItemLeishmania carbon metabolism in the macrophage phagolysosome- feast or famine?(F1000 Research Ltd, 2015)A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments. How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania.
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ItemInduction of a Stringent Metabolic Response in Intracellular Stages of Leishmania mexicana Leads to Increased Dependence on Mitochondrial Metabolism(PUBLIC LIBRARY SCIENCE, 2014-01)Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using (13)C-stable isotope resolved metabolomics and (2)H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.
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ItemIntracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages(PUBLIC LIBRARY SCIENCE, 2015-09)Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.
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ItemCharacterization of Metabolically Quiescent Leishmania Parasites in Murine Lesions Using Heavy Water Labeling(PUBLIC LIBRARY SCIENCE, 2015-02)Information on the growth rate and metabolism of microbial pathogens that cause long-term chronic infections is limited, reflecting the absence of suitable tools for measuring these parameters in vivo. Here, we have measured the replication and physiological state of Leishmania mexicana parasites in murine inflammatory lesions using 2H2O labeling. Infected BALB/c mice were labeled with 2H2O for up to 4 months, and the turnover of parasite DNA, RNA, protein and membrane lipids estimated from the rate of deuterium enrichment in constituent pentose sugars, amino acids, and fatty acids, respectively. We show that the replication rate of parasite stages in these tissues is very slow (doubling time of ~12 days), but remarkably constant throughout lesion development. Lesion parasites also exhibit markedly lower rates of RNA synthesis, protein turnover and membrane lipid synthesis than parasite stages isolated from ex vivo infected macrophages or cultured in vitro, suggesting that formation of lesions induces parasites to enter a semi-quiescent physiological state. Significantly, the determined parasite growth rate accounts for the overall increase in parasite burden indicating that parasite death and turnover of infected host cells in these lesions is minimal. We propose that the Leishmania response to lesion formation is an important adaptive strategy that minimizes macrophage activation, providing a permissive environment that supports progressive expansion of parasite burden. This labeling approach can be used to measure the dynamics of other host-microbe interactions in situ.
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ItemMembrane targeting of the small myristoylated protein 2 (SMP-2) in Leishmania major(ELSEVIER SCIENCE BV, 2013-07)Leishmania parasites express three highly conserved small myristoylated proteins (SMPs) that are targeted to distinct membranes. SMP-1 is exclusively found in the flagellum, depending on myristoylation and palmitoylation. In contrast, monoacylated SMP-2 and SMP-4 are localized to the flagellar pocket and plasma membrane, respectively. Here, we demonstrate that unlike SMP-4, SMP-2 resides in detergent resistant membranes, but can be readily solubilized in the presence of high concentrations of salt. We provide evidence that in detergent resistant membranes, SMP-2 forms high molecular weight complexes in vivo. Association with detergent resistant membranes was abrogated in the presence of a C-terminal tag suggesting acylation independent targeting signals. In addition, the N-terminal region of SMP-2 contains sufficient information for membrane targeting, as a GFP-chimera localizes to the flagellar pocket. Thus while the core sequences of the SMPs are highly conserved, individual members have evolved different mechanisms for their diverse membrane localization.
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ItemLeishmania major Methionine Sulfoxide Reductase A Is Required for Resistance to Oxidative Stress and Efficient Replication in Macrophages(PUBLIC LIBRARY SCIENCE, 2013-02-20)Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The ΔmsrA mutant exhibited increased sensitivity to H(2)O(2) compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H(2)O(2) and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the ΔmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.