Fig 1.
Overview of comparative transcriptomics analysis of T. brucei and T. congolense, isolated from ex vivo and in vitro conditions.
RNAseq data from T. congolense (IL3000) and T. brucei (STIB 247) in both in vitro and ex vivo (from mouse infections) conditions were aligned to the species’ respective reference genomes and read counts were normalised by the transcripts per million (TPM) method. To directly compare the species, a pseudogenome was generated using the Orthofinder tool [57]. TPM values from the 4 sample groups were plotted against each other to analyse correlation between conditions (A and B) and between species in the same conditions (C and D). Correlation was assessed using both Pearson correlation (Pearson’s r) and Spearman’s rank correlation coefficients.
Fig 2.
Analysis of supernatant metabolites after T. congolense culture.
A heatmap covering the 80 putative medium components judged to be significantly altered after 56 hours of in vitro cell culture containing T. congolense strain IL3000, as calculated by a one-way repeated measures ANOVA (P < 0.05). Peak abundances were log transformed and mean centred and metabolites were clustered based on Pearson correlation. Two clusters of interest were identified, which are shown in a larger format on the right. Metabolites in the top cluster were observed to increase significantly over time, whilst those in the bottom cluster decreased. Metabolite names followed by {*} were matched to an authentic standard and all other identifications are putative based on mass, retention time and formula. B) Comparison of metabolite changes in medium supernatants after 56 hours between T. brucei [60] and T. congolense (S3 Table). Relative changes in metabolite abundance were calculated as log2 fold change of 56 h vs 0 h and metabolites exhibiting differences between the species are listed next to the figure.
Fig 3.
Energy metabolism in T. congolense.
A-E) Supernatant metabolomics analysis of metabolites involved in glycolytic metabolism in T. congolense. Grey bars indicate a negative medium control incubated for 56 hours. F) A commercial kit was used to measure acetate concentration during T. congolense culture, with supernatant samples analysed at the same time points as the supernatant metabolomics experiment. G) A simplified overview of the glycolytic pathway. Numbers refer to the following proteins: 1, hexokinase; 2, glucose 6-phosphate isomerase; 3a, phosphofructokinase; 3b, fructose-1,6-bisphosphatase; 4, aldolase; 5, triosephosphate isomerase; 6, glycerol 3-phosphate dehydrogenase; 7, glycerol kinase; 8, glyceraldehyde 3-phosphate dehydrogenase; 9, phosphoglycerate kinase; 10, phosphoglycerate mutase; 11, enolase; 12, phosphenolpyruvate carboxykinase; 13, malate dehydrogenase; 14, fumarate hydratase; 15, NADH-dependent fumarate reductase; 16, pyruvate kinase; 17, alanine aminotransferase; 18, pyruvate dehydrogenase complex; 19, acetate:succinate CoA-transferase and acetyl-CoA thioesterase. H) Tracing glucose derived carbon usage through glycolytic metabolism. T. congolense were incubated with a 50:50 mix of 12C-D-glucose:13C-U-D-glucose before cell pellets were isolated for metabolomics analysis. Results were compared to those generated in T. brucei by Creek and colleagues [45]. Colours indicate the number of 13C atoms in each metabolite. I) Comparative analysis of transcript level activity of glycolysis in T. brucei and T. congolense from both in vitro and ex vivo conditions. Gene IDs: HK1 & 2, hexokinase, TbTc_0341; GPI, glucose 6-phosphate isomerase, TbTc_1840; PFK, phosphofructokinase, TbTc_1399; ALDA, aldolase, TbTc_0358; TPI, Triosephosphate isomerase, TbTc_1075; GPDH, glycerol 3-phosphate dehydrogenase, TbTc_2722; GK, glycerol kinase, TbTc_0392; GAPDH, glyceraldehyde 3-phosphate dehydrogenase, TbTc_0377; PGK, phosphoglycerate kinase, TbTc_6030; PGKA, phosphoglycerate kinase A, TbTc_0241; PGKB/C, phosphoglycerate kinase B & C, TbTc_0240; PGM, phosphoglycerate mutase, TbTc_5039; ENO1, enolase, TbTc_0465; ENO, putative, enolase, putative, TbTc_3614; PK1, pyruvate kinase 1, TbTc_0372; FBPase, fructose-1,6-bisphosphatase, TbTc_1967; PEPCK, phosphoenolpyrvuate carboxykinase, TbTc_0348; gMDH, glycosomal malate dehydrogenase, TbTc_0642; FH, fumarate hydratase, TbTc_0242; Frd, NADH-dependent fumarate reductase, TbTc_0141; PPDK, pyruvate phosphate dikinase, TbTc_1304; AAT, alanine aminotransferase, TbTc_0675; PDH E1α, pyruvate dehydrogenase E1 alpha subunit, TbTc_4169; PDH E1β, pyruvate dehydrogenase E1 beta subunit, TbTc_5437.
Fig 4.
In vitro analysis of glycolytic metabolism.
A) T. congolense remains viable in reduced glucose concentrations. A growth defect was only observed when glucose concentrations were reduced to <2 mM. B) Supplementation with increased concentrations of 2-deoxy-D-glucose leads to T. congolense cell death (red dotted line indicates detection limit by haemocytometer). C) Analysis of growth in the presence and absence of N-acetyl-D-glucosamine. Parasites were cultured in SCM-6 supplemented with 10 mM or 2 mM glucose in the presence or absence of 60 mM GlcNAc and density monitored by haemocytometer every 24 hours. D) Knock-down of the entire glucose transporter (TcoHT) array does not affect in vitro cell viability. RNAi was induced in three independent clones by the addition of 1 μg/mL tetracycline (Tet), and cell densities of induced and uninduced cells were monitored daily. E) Normalised TcoHT mRNA abundance over time after RNAi induction. F) Changes in glucose uptake in RNAi-induced cells were detected via an enzyme-linked luminescence assay coupled to 2-deoxy-D-glucose uptake over a period of 30 minutes. The assay was carried out 72-hours post-induction. Of the three RNAi lines, 2 showed a significant reduction in glucose uptake capability (Student’s T-test, *P < 0.05; ***P < 0.001).
Fig 5.
Nucleotide metabolism in T. congolense.
Supernatant analysis of T. congolense in vitro cultures showing changes in abundance of D-ribose (A), guanine (B), xanthine (C) and inosine (D) over 56 hours. Grey bar indicates a negative medium control group E) Simplified overview of purine salvage and synthesis in trypanosomatids (adapted from [148]). Numbers indicate the following enzymes: 1, APRT; 2, AD; 3, HGPRT; 4, IMPD; 5, HGXPRT; 6, GMPR; 7, GMPS; 8, HGPRT. Red cross indicates guanine deaminase, which is not encoded/annotated in the T. congolense genome (based on current assembly). F) Comparison of glucose-derived purine carbon labelling in T. congolense and T. brucei [45]. Colours indicate the number of 13C atoms in each metabolite. G) Comparative RNAseq analysis of T. congolense and T. brucei under both in vitro and ex vivo conditions. Gene IDs from top to bottom: PWY0-162 (pyrimidine biosynthesis): PYR1A-B, glutamine hydrolysing carbomoyl phosphate synthase, TbTc_1631; DHODH, dihydroorotate dehydrogenase (fumarate), TbTc_0620; CTPS, cytidine triphosphate synthase, TbTc_0920; PYR3, dihydroorotase, TbTc_3801; PYR2, aspartate carbamoyltransferase, TbTc_1630; CMF40a, nucleoside diphosphate kinase, TbTc_5784; OMPDC/OPRT, orotidine-5-monophosphate decarboxylase/orotate phosphoribosyltransferase, TbTc_0735. PWY0-163 (pyrimidine salvage): CMF40a, nucleoside diphosphate kinase, TbTc_5784; NDPK3, nucleoside diphosphate kinase 3, TbTc_2560; NDPK, nucleoside diphosphate kinase, TbTc_0593; CDA, cytidine deaminase, TbTc_3318; UPRT, uracil phosphoribosyltransferase, TbTc_4220; UP, uridine phosphorylase, TbTc_5794. P121-PWY (adenine/adenosine salvage): ADSL, adenylosuccinate lyase, TbTc_1986; APRT-2, glycosomal adenine phosphoribosyltransferase, TbTc_5918; GMPR, GMP reductase, TbTc_4627; IMPDH1, inosine-5’-monophosphate dehydrogenase, TbTc_1648; HGXPRT, hypoxanthine-guanine-xanthine phosphoribosyltransferase, TbTc_3696; APRT-1, cytosolic adenine phosphoribosyltransferase, TbTc_3522; HGPRT, hypoxanthine-guanine phosphoribosyltransferase, TbTc_0726; ADSS, adenylosuccinate synthetase, TbTc_1142.
Fig 6.
Amino acid metabolism in T. congolense IL3000.
A-C) Analysis of indicated amino acids in T. congolense IL3000 culture supernatants over a 56 h time course. Grey bars indicate a negative medium control group. D-F) Growth curves in SCM-6 excluding one amino acid at a time, to determine those essential to T. congolense viability. In each experiment, full SCM-6 was used as a positive control. Legends indicate which amino acid was removed in each experiment. G) Growth analysis of SCM-6 and SCM-7, the latter containing only amino acids deemed essential, compared to HMI-93 [124]. H) Simplified map of intracellular glutamine metabolism. Numbers refer to the following enzymes: 1, glutaminase; 2, glutamate decarboxylase; 3, 4-aminobutyrate aminotransferase; 4, succinate semialdehyde dehydrogenase; 5, glutamate dehydrogenase; 6, 2-oxoglutarate dehydrogenase; 7, Succinyl-CoA synthetase; 8, isocitrate dehydrogenase; 9 & 10, aconitase. I) Carbon utilisation from L-glutamine was analysed in T. congolense (100% 13C-U-L-glutamine) and compared to that in T. brucei (50:50 ratio of L-glutamine and 13C-U-L-glutamine) [89].
Fig 7.
Fatty acid metabolism in T. congolense.
A) Glucose-derived 13C carbon labelling of saturated fatty acids in T. congolense and T. brucei [45]. Colours correspond to the number of 13C labels detected in each metabolite. B) L-threonine-derived saturated fatty acid 13C labelling in T. congolense. Fatty acid systematic names and numbers: lauric acid: dodecanoic acid, C12:0; myristic acid: tetradecanoic acid, C14:0; palmitic acid: hexadecanoic acid, C16:0; nonadecyclic acid: nonadecanoic acid, C19:0. C) Transcriptomics analysis of acetate and lipid metabolism. Gene names and IDs: Acetate metabolism (PWY1V8-8): AKCT, 2-amino-3-ketobutyrate-CoA ligase, TbTc_6236; TDH, L-threonine 3-dehydrogenase, TbTc_5991; AceCS, acetyl-CoA synthetase, TbTc_0318; PYK1, pyruvate kinase, TbTc_0372; PDHe3, pyruvate dehydrogenase E3, TbTc_4765; PDHe1β, pyruvate dehydrogenase E1 β subunit, TbTc_5437; PPDK, pyruvate phosphate dikinase, TbTc_1304; SCSα, succinyl-CoA synthetase α subunit, TbTc_0813; PDHe1α, pyruvate dehydrogenase E1 α subunit, TbTc_4169; ACH, acetyl-CoA hydrolase/thioesterase, TbTc_5515; PDHe2, dihydrolipoamide acetyltransferase, TbTc_1015. Fatty acid biosynthesis (PWY0-881): ACC, acetyl-CoA carboxylase, TbTc_0754; BKS, β-ketoacyl synthase, TbTc_3372; BKR, β-ketoacyl-ACP reductase, TbTc_1241. Sterol metabolism (PWY1V8-3): SPPS, solanesyl-diphosphate synthase, TbTc_3025; SQase, squalene synthase, TbTc_2577; CYP51A1, lanosterol 14α demethylase, TbTc_4837; SMT, sterol 24-c methyltransferase, TbTc_0387; LSS, lanosteral synthase, TbTc_4540; MVK, mevalonate kinase, TbTc_3761; FPPS, farnesyl pyrophosphate synthase, TbTc_5375; HMGCL, hydroxymethylglutaryl-CoA lyase, TbTc_6160; SM, squalene monooxygenase, TbTc_3357; MDD, mevalonate diphosphate decarboxylase, TbTc_0546; IDI, isopentenyl-diphosphate delta-isomerase, TbTc_1099; PTase, prenyltransferase, TbTc_1352; GGTase-IIβ, geranylgeranyl transferase type II β subunit, TbTc_0680; SCP2, 3-ketoacyl-CoA thiolase, TbTc_4024; PMVK, phosphomevalonate kinase, TbTc_3039; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase, TbTc_3189. Fatty acid oxidation (FAO-PWY): ECH, enoyl-CoA hydratase, TbTc_3283; ACS3/ACS4, fatty acyl-CoA synthetase 3 & 4, TbTc_0101; ACS2, fatty acyl-CoA synthetase 2, TbTc_0102; LACS5, fatty acyl-CoA synthetase, TbTc_0099; ACSL_0688, long-chain-fatty-acid-CoA ligase, TbTc_0688; ACSL_2381, long-chain-fatty-acid-CoA ligase, TbTc_2381; ACS1, fatty acyl-CoA synthetase 1, TbTc_0100; TFEα1, enoyl-CoA hydratase/enoyl-CoA isomerase, TbTc_3362; ECI_4184, 3,2-trans-enoyl-CoA isomerase, TbTc_4184; SCP2, 3-ketoacyl-CoA thiolase, TbTc_4024; ECI_0360, 3,2-trans-enoyl-CoA isomerase, TbTc_0360; ACAD, acyl-CoA dehydrogenase, TbTc_4954.
Table 1.
Comparative analysis of sensitivity to metabolic inhibitors in T. congolense and T. brucei.
Abbreviations: FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; SHAM, salicylhydroxamic acid; TAO, trypanosome alternative oxidase; AceCS, acetyl-CoA synthetase.
Fig 8.
Summary of T. congolense and T. brucei in vitro transcriptome.
Log2 fold change (T. congolense/T.brucei) was calculated for each gene. Dashed lines represent transport processes. Genes: 1, hexose transporters, TbTc_0095; 2, hexokinase, TbTc_0341; 3, glucose-6-phosphate isomerase, TbTc_1840; 4, phosphofructokinase, TbTc_1399; 5, fructose-1,6-bisphosphatase, TbTc_1967; 6, aldolase, TbTc_0358; 7, triosephosphate isomerase, TbTc_1075; 8, glycerol-3-phosphate dehydrogenase, TbTc_2722; 9, glycerol kinase, TbTc_0392; 10, glyceraldehyde 3-phosphate dehydrogenase, TbTc_0377; 11, phosphoglycerate kinase, TbTc_0240; 12, phosphoglycerate mutase, TbTc_5039; 13, enolase, TbTc_0465; 14, pyruvate kinase 1, TbTc_0372; 15, alanine aminotransferase, TbTc_0675; 16, pyruvate phosphate dikinase, TbTc_1304; 17, Phosphoenolpyruvate carboxykinase, TbTc_0348; 18, glycosomal malate dehydrogenase, TbTc_0642; 19, glycosomal fumarate hydratase, TbTc_0242; 20, glycosomal NADH-dependent fumarate reductase, TbTc_0140; 21, glucose-6-phosphate dehydrogenase, TbTc_0931; 22, 6-phosphogluconolactonase, TbTc_4165; 23, 6-phosphogluconate dehydrogenase, TbTc_2025; 24, ribulose-5-phosphate epimerase, TbTc_4356; 25, ribose 5-phosphate isomerase, TbTc_3090; 26, transketolase, TbTc_1701; 27, transaldolase, TbTc_1823; 28, ribokinase, TbTc_5212; 29, malic enzyme, TbTc_0296; 30, Mitochondrial pyruvate carrier 2, TbTc_2668; 31, FAD-dependent glycerol-3-phosphate dehydrogenase, TbTc_2282; 32, NADH dehydrogenase (NDH2), TbTc_5033; 33, Alternative oxidase, TbTc_6589; 34, mitochondrial fumarate hydratase, TbTc_0243; 35, mitochondrial NADH-dependent fumarate reductase, TbTc_0141; 36, mitochondrial malate dehydrogenase, TbTc_0256; 37, citrate synthase, TbTc_0486; 38, aconitase, TbTc_5765; 39, isocitrate dehydrogenase, TbTc_0510; 40, 2-oxoglutarate dehydrogenase E1 component, TbTc_2864; 41, 2-oxoglutarate dehydrogenase E1 component, TbTc_3111; 42, 2-oxoglutarate dehydrogenase E2 component, TbTc_3057; 43, succinyl-CoA synthetase α, TbTc_0813; 44, succinyl-CoA ligase β, TbTc_3392; 45, glutamine synthetase, TbTc_2226; 46, glutamate dehydrogenase, TbTc_0872; 47, pyruvate dehydrogenase E1 α subunit, TbTc_4169; 48, pyruvate dehydrogenase E1 β subunit, TbTc_5437; 49, dihydrolipoamide acetyltransferase, TbTc_1015; 50, pyruvate dehydrogenase complex E3, TbTc_4765; 51, L-threonine 3-dehydrogenase, TbTc_5991; 52, 2-amino-3-ketobutyrate coenzyme A ligase, TbTc_6236; 53, Acetyl-CoA hydrolase (ACH), TbTc_5515; 54, Succinyl-CoA:3-ketoacid coenzyme A transferase (ASCT), TbTc_0236; 55, Acyl carrier protein, TbTc_5262; 56, beta-ketoacyl-ACP synthase, TbTc_3372; 57, beta-ketoacyl-ACP reductase, TbTc_1241; 58, Trans-2-enoyl-ACP reductase 1, TbTc_5269; 59, acetyl-CoA synthetase, TbTc_0318; 60, acetyl-CoA carboxylase, TbTc_0754; 61, Fatty acid elongase (ELO1), TbTc_0159; 62, Fatty acid elongase (ELO2), TbTc_1882; 63, Fatty acid elongase (ELO3), TbTc_0235; 64, elongation of very long chain fatty acids protein (ELO4), TbTc_0737; 65, aspartate aminotransferase, TbTc_0799; 66, aspartate carbamoyltransferase, TbTc_1630; 67, dihydroorotase, TbTc_3801; 68, dihydroorotate dehydrogenase, TbTc_0620; 69, orotidine-5-phosphate decarboxylase/orotate phosphoribosyltransferase, TbTc_0735; 70, uracil phosphoribosyltransferase, TbTc_4220; 71, Adenine phosphoribosyltransferase (APRT-2), TbTc_3522; 72, inosine-adenosine-guanosine-nucleoside hydrolase, TbTc_4998; 73, adenosine kinase, TbTc_1024; 74, AMP deaminase, TbTc_5808; 75, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), TbTc_0726; 76, inosine-guanine nucleoside hydrolase, TbTc_0808; 77, inosine-5’-monophosphate dehydrogenase, TbTc_1648; 78, Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), TbTc_3696; 79, GMP reductase, TbTc_4627; 80, GMP synthase, TbTc_1452. Abbreviations: PUFA, polyunsaturated fatty acid.