Mice infection with <i>Δfbp</i>.<i>sbp</i>.<i>tal</i> cell line.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.</div

Standards for LC/MS metabolomics.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.</div

Generation and growth analysis of Δ<i>fbp</i>.<i>sbp</i> strains.

A–The scheme shows glycolysis, gluconeogenesis, and the pentose phosphate pathway in BSF T. brucei with highlighted FBPase, PFK, and TAL. Missing reactions of transketolase are depicted by grey arrows. B–A scheme for the Cas9 editing method by transient transfection. The templates for an sgRNA and for an antibiotic resistance cassette were transfected simultaneously, which resulted in replacement of both alleles for FBPase in the first step, and for SBPase in the second step. Fig 1B was created in BioRender. C–Validation of the Δfbp.sbp cell line by PCR, shows parts of ORFs for FBPase and SBPase amplified in the parental 2T1T7.Cas9 cell line, but absent from Δfbp.sbp. D—Validation of the Δfbp.sbp cell line by western blot. Only the parental cell line shows signal for FBPase (whole cell lysates) and SBPase (organellar fractions), *—cross-reacting protein, gel loading–fluorescent protein detection on a TGX gel. E–Growth curves of two independent clones of Δfbp.sbp show no defect in the standard HMI-11 medium. Growth curves in the CMM medium show a mild growth defect of the Δfbp.sbp clones and higher variability.</p

Sequences of DNA primers used in the study.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.</div

Octulose 8-phosphate (O8P) as detected in the <i>Δtal</i> cell lines by LC-MS metabolomics.

13C indicates medium supplemented with 13C-glycerol as the only carbon source. (TIF)</p

LC-MS metabolomics in HMI-11 medium depleted of glucose, but supplemented with ¹³C₃-glycerol.

2T1 13C –parental cell line in medium with 13C-glycerol, PFKRNAi–non-induced PFK RNAi cell lines in medium with 13C-glycerol, PFKRNAi + Tet–PFK RNAi induced for 24 h in medium with 13C-glycerol, Δfbp.sbp/PFK–non-induced Δfbp.sbp/RNAiPFK cell line in medium with 13C-glycerol, Δfbp.sbp/PFK + Tet—Δfbp.sbp/RNAiPFK cell line induced for 24 h in medium with 13C-glycerol. (TIF)</p

PFK knockdown causes a severe growth defect, but only mild changes in the metabolome.

A–Growth curves of PFKRNAi and in Δfbp.sbp/RNAiPFK show a severe growth defect after RNAi. Levels of PFK mRNA are shown as detected by qRT-PCR, compared to non-induced cells, and normalised to 18S, 24 h tetracycline induction (insets). B–LC-MS metabolomics in HMI-11 medium depleted of glucose, and supplemented with 13C3-glycerol. The proportion of the C13 label incorporation relative to the total amount is indicated. G3P –glycerol 3-phosphate, F6P –fructose 6-phosphate, F1,6bP–fructose 1,6-bisphosphate. 2T1 13C –parental cell line in medium with 13C-glycerol, PFKRNAi–non-induced PFK RNAi cell lines in medium with 13C-glycerol, PFKRNAi + Tet–PFK RNAi induced for 24 h in medium with 13C-glycerol, Δfbp.sbp/PFK–non-induced Δfbp.sbp/RNAiPFK cell line in medium with 13C-glycerol, Δfbp.sbp/PFK + Tet—Δfbp.sbp/RNAiPFK cell line induced for 24 h in medium with 13C-glycerol. C–Same as in B), but relative changes in the total levels of metabolites are depicted. P5P –pentose 5-phosphates, O8P –octulose 8-phosphate, S7P –sedoheptulose 7-phosphate.</p

Gluconeogenic activity is maintained in Δ<i>fbp</i>.<i>sbp</i> strains.

A–Changes in Δfbp.sbp cells grown in CMM and subjected to LC-MS metabolomics when compared to the parental cell line. The most changed metabolites are S7P, S1,7bP, and phosphothreonine. The plot includes 87 metabolites from glycolysis, PPP, TCA cycle or amino acid metabolism. B–Sedoheptulose 1,7-bisphosphate (S1,7bP), sedoheptulose 7-phosphate (S7P), fructose 1,6-bisphosphate (F1,6bP), and fructose 6-phosphate (F6P) as detected in the parental and Δfbp.sbp cells grown in CMM and subjected to LC-MS metabolomics. C—Δfbp.sbp cells were grown in HMI-11 supplemented with U-13C3-glycerol and analysed by LC-MS metabolomics, showing that 13C from glycerol is incorporated into GNG products. H6P –hexose 6-phosphates. D—Δfbp.sbp cells were grown in CMM supplemented with glucose, or U-13C3-glycerol and analysed by LC-MS metabolomics. G6P –glucose 6-phosphate.</p

Deletion of TAL does not decrease GNG flux.

A—The novel pathway as suggested by Hannaert [25]: Erythose 4-phosphate (E4P) is condensated with dihydroxyacetone phosphate (DHAP) by fructose-1,6-bisphosphate aldolase into S1,7bP, which is dephosphorylated by SBPase into S7P. That is used as a substrate by transaldolase, together with glyceraldehyde 3-phosphate (GA3P), and converted into F6P and E4P, which can enter another cycle. B–PCR detection of a 500-bp product from the TAL ORF, proving deletion of the gene (top panel). PCR with oligos annealing to to the UTRs of the TAL gene, amplifying the TAL gene in the parental cell line (1,402 bp), replaced by phleomycin resistance gene (775 bp) in Δfbp.sbp.tal, and both phleomycin resistance and puromycin N-acetyltransferase (1,006 bp) in Δtal (bottom panel). C–Growth curves of the parental (2T1T7-Cas9), Δtal, and Δfbp.sbp.tal cell lines in HMI-11 medium supplemented with glucose, or glycerol only (and 10% FBS). D—LC-MS metabolomics in CMM supplemented with 5 mM 13C3-glycerol. G6P –glucose 6-phosphate, F1,6bP—fructose 1,6-bisphosphate, S7P - sedoheptulose 7-phosphate. 2T1 –parental cell line in medium with glucose, 2T1 13C –parental cell line in medium with 13C-glycerol, Δtal13C –Δtal cell line in medium with 13C-glycerol, Δfbp.sbp.tal—Δfbp.sbp.tal cell line in medium with glucose, Δfbp.sbp.tal13C - Δfbp.sbp.tal cell line in medium with 13C-glycerol.</p

PFK and FBPase co-localise in glycosomes.

A–PCR validation of TyPFK clones. B–Western blot validation of the TyPFK cell line. C–Immunofluorescence assay with TyPFK cell line, α-Ty, and α-FBPase staining. Cells were cultured in CMM with different carbon sources (glucose, glycerol, or both), but it had no effect on co-localisation of PFK and FBPase.</p