Comparative study of amino acids utilisation in Leishmania mexicana

Critical metabolic steps of the central carbon metabolism pathways have been of primary focus for the discovery of novel drug targets against Leishmaniasis, a neglected tropical disease caused by Leishmania. It is known that this particular protozoan parasite also relies on exogenous amino acids; however, comparative studies between exogenous amino acids utilisation and their essentiality for parasite growth and metabolism were often less emphasised. In this thesis, a novel approach was used to apply metabolomics techniques in a newly developed amino acids rich chemically defined medium for in vitro culture of Leishmania promastigotes. The chemically defined medium allowed for controlled customisable environment for deeper understanding of amino acids metabolism and parasite growth. A previously reported semi-defined growth medium (REIX (Steiger and Black, 1980)) was selected as a starting point. Undefined components of REIX were omitted and the composition of the resulting defined medium was varied through the addition, individually or in groups of amino acids and trace components (including magnesium, calcium, zinc, iron salts, biopterin, folate, riboflavin and lipoic acid). Growth assays were performed to identify a medium composition, referred to as Nayak medium, that supported robust growth over serial passages, at a similar rate to a conventional, serum supplemented rich growth medium (HOMEM (Berens et al., 1976)). Subsequently, the importance of individual amino acids was studied by systematic analysis in media containing equimolar quantities of each proteogenic amino acid, bar one (termed “amino acid knock out media”). Growth rate, concentration dependent growth, cell body length, total cell protein and acetate secretion were all recorded. Based on these results, it was found that exogenous supply of L-Tryptophan, L-Phenylalanine, L-Arginine, L-Lysine, L-Leucine and L-Valine as most critical for viability of promastigotes. Hence, it was of interest to further investigate the intracellular composition of the other 14 amino acids and their anabolic and catabolic pathways within L. mexicana promastigotes. Comparative investigation of intracellular amino acid metabolism was conducted using a metabolomic finger-printing technique in log phase L. mexicana promastigotes cultured in defined medium. Previous studies on amino acid pathways were mostly based on genome annotation or reductionist experimental approaches, however, network mapping of metabolomics data as shown in Chapter 5 allowed to understand new insights about compensatory amino acid metabolic pathways, not predicted before. The 45 known components of the defined medium allowed for easier distinction from ~400 metabolites of the intracellular samples. The free amino acids pool showed increased abundance of L-Alanine, L-Glycine, L-Proline, L-Histidine, L-Asparagine, L-Threonine and L-Tyrosine in the intracellular metabolome which partly explains the dispensability of these amino acids in culture media as observed in single amino acid knock out experiments. Approximately 64 metabolites, putatively identified as related to amino acid metabolism were analysed by pathway mapping. This showed a unique utilisation pattern amongst all amino acids following a two-step transamination and a decarboxylation with significant NADH production. The downstream intermediates were secreted into the environment, rather than elaborate degradation pathways as followed within the mammalian systems. Validation of the observed secretion pattern of amino acid degradation was conducted using metabolic foot-printing technique at six different time points, which provided data on relative quantification of metabolite uptake and secretion from the culture media at different growth stages. About ~200 metabolites were putatively identified from across the replicates in the dataset collected over a growth period of 9 days in both the positive and negative ionisation mode and only ~70 metabolites could be identified with authentic standards for individual mass/charge ratio and retention time comparison. Depletion profiling of the culture media showed that L-Tryptophan, L-Aspartate, L-Glutamate, L-Arginine, L-Methionine and L-serine were continuously utilised with more than 50% consumed over the 9-day growth period. Amino acids such as L-Leucine, L-Threonine, L-Valine, L-Phenylalanine, L-Tyrosine and L-Lysine were partially utilised, while L-Glycine, L-Glutamine, L-Asparagine, L-Proline and L-Alanine were continuously secreted during the growth period under conditions tested. Overall, ~25 % of the total metabolites were found depleted from the medium, ~15% not changed significantly, 20% un-identified metabolites and interestingly, about ~40% of metabolites were significantly enriched in the medium. Systematic analysis of the 40% secreted small molecules secreted in the culture media constituted the exometabolome. The proportion of keto acids exceeded the other constituents of the exometabolome, confirming the partial degradation and overflow metabolism. Hence, the possibility that keto acids could also contribute to virulence was speculated. Early results from various biochemical analysis allowed new insights about amino acid derived enol lactones, especially the aromatic pyruvates to support the establishment of parasitism, with macrophage cells as in vitro experimental model. However, targeted molecular biology studies are required to further understand the role of exometabolome in parasitism; especially by inhibition of the Leishmania enzymes L-Tryptophan transaminase or phenyl pyruvate dehydrogenase as they are non-homologous to mammalian systems. In summary, this study has shown simple, customisable platform for in vitro culture of Leishmania promastigotes in defined medium, intracellular and exometabolome of Leishmania adaptable for various studies. L-Tryptophan and L-Phenylalanine were found as the most critical exogenous amino acids for parasite viability and their analogues inhibited parasite growth. Hence, warrants further studies for their effectiveness as drugs in the field. Also, Leishmania exometabolome could serve as diagnostic signature for species identification, taxonomical comparisons, virulence patterns, physiological comparisons within varied nutrient systems.