Quantitative proteomic and metabolomic characterization of glucose transporter mutant promastigotes of Leishmania mexicana

[摘要] Parasitic protozoa of the genus Leishmania possess a highly adaptable metabolic system which ensures their survival in two significantly contrasting in terms of metabolism hosts. A critical component of leishmanial metabolic machinery is the central carbon metabolism which encompasses a major fraction of metabolites and constitutively expressed enzymes whose localization, function, and manner of regulation in response to changes in nutrient levels in the different host niches, however, remain unclear. In a quest to elucidate how the Leishmania parasites overcome conditions with diminished levels of the primary nutrient D-glucose, a null-mutant cell line that was deprived of the ability to acquire the hexose by genetic ablation of three related glucose transporters was created. As previously shown, the glucose transport deficiency in the null-mutant Leishmania mexicana (Δlmgt) is associated with reduced growth of the promastigote form in an axenic culture and the sand fly vector, no detectable utilization of the sugars D-glucose, D-fructose, D-mannose, D-galactose, and D-ribose, reduced biosynthesis of sugar-containing glycoconjugates and virulence factors, increased sensitivity to nutrient starvation, elevated temperatures, and oxidative stress, and dramatically reduced levels of growth and parasitemia of the amastigote form in an axenic culture and macrophages. Considering all phenotypic characteristics observed previously, the main aims of this study were to determine whether the Δlmgt promastigotes use alternative carbon and energy sources, to investigate which pathways are altered as a result of possible utilization of alternative carbon sources, and to illustrate, as comprehensively as possible, the molecular events behind the changes in the central carbon metabolism in the Δlmgt promastigotes. For that purpose, a number of proteomic and metabolomic techniques were applied to identify and quantify modulations in protein and metabolite abundance in the Δlmgt promastigotes. The results revealed that the main energy and carbon sources for the Δlmgt promastigotes appear to be amino acids, with the tricarboxylic acid cycle playing a central role in amino acid catabolism. Furthermore, glycolysis/gluconeogenesis, pentose phosphate pathway, mannose metabolism, purine salvage pathway, and β-oxidation of fatty acids were also among the pathways affected by the glucose transporter incapacity of the Δlmgt promastigotes. Glycolysis/gluconeogenesis was shown to operate in a gluconeogenic mode and to be used for the synthesis of hexose phosphates. The oxidative phase of the pentose phosphate pathway and the glutathione metabolism were found to be down-regulated in the Δlmgt promastigotes, which is believed to be the main reason behind the increased sensitivity of these parasites to oxidative stress. Mannose metabolism, which provides activated substrates for the synthesis of the variety of leishmanial secreted and membrane-bound glycoconjugates and the carbohydrate reserve material and virulence factor mannogen, was also down-regulated and that finding corroborated with the previous observation of reduced glycoconjugate and mannogen synthesis in the Δlmgt promastigotes. Pyrimidine metabolism was not significantly modulated but the purine salvage pathway was down-regulated in the Δlmgt promastigotes. Nonetheless, the Δlmgt promastigotes appear to salvage ribose from nucleotide degradation and recycle it for the synthesis of new nucleotides. β-Oxidation of fatty acids was also decreased in the Δlmgt promastigotes, suggesting that lipids are not a major source of energy for these cells. Altogether, the data showed that the Δlmgt promastigotes i/ decrease high energy-consuming processes such as DNA, RNA, and protein synthesis, ii/ rely heavily on amino acid catabolism via the tricarboxylic acid cycle for energy generation, iii/ use alternative carbon sources for the production of biosynthetic precursors, iv/ have reduced capabilities to synthesize key metabolites such as sugar phosphates and sugar-containing macromolecules, and v/ are characterised with impaired regeneration of the antioxidant defence system and decreased production of virulence factors which appear to determine the susceptibility of these organisms to oxidative stress.

[发布机构] University:University of Glasgow;Department:Institute of Infection Immunity and Inflammation

[关键词] Leishmania, proteomics, metabolomics.