Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei

Julie Kovarova, Rupa Nagar, Joana Correia Faria, Michael Ferguson, Michael P. Barrett, David Horn (Lead / Corresponding author)

Research output: Contribution to journalArticle

Abstract

Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.
LanguageEnglish
Article numbere1007475
Pages1-22
Number of pages22
JournalPLoS Pathogens
Volume14
Issue number12
DOIs
Publication statusPublished - 27 Dec 2018

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Trypanosoma brucei brucei
Gluconeogenesis
Glycerol
Trypanosomiasis
Monosaccharide Transport Proteins
Adenosine Triphosphate
Glycolysis
Glucose
Parasites
Growth
Cell Membrane
Neglected Diseases
Fructose-Bisphosphatase
Pentose Phosphate Pathway
Gene Knockout Techniques
Hexoses
Metabolomics
Citric Acid Cycle
Facilitative Glucose Transport Proteins
Membrane Glycoproteins

Cite this

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title = "Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei",
abstract = "Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.",
author = "Julie Kovarova and Rupa Nagar and {Correia Faria}, Joana and Michael Ferguson and Barrett, {Michael P.} and David Horn",
note = "The work was funded by Wellcome Trust (https://wellcome.ac.uk/) Investigator Awards to DH (100320/Z/12/Z) and MAJF (101842/Z13/Z), with additional support from Wellcome Trust Centre Awards to Dundee (203134/Z/16/Z) and Glasgow (104111/Z/14/Z).",
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T1 - Gluconeogenesis using glycerol as a substrate in bloodstream-form Trypanosoma brucei

AU - Kovarova, Julie

AU - Nagar, Rupa

AU - Correia Faria, Joana

AU - Ferguson, Michael

AU - Barrett, Michael P.

AU - Horn, David

N1 - The work was funded by Wellcome Trust (https://wellcome.ac.uk/) Investigator Awards to DH (100320/Z/12/Z) and MAJF (101842/Z13/Z), with additional support from Wellcome Trust Centre Awards to Dundee (203134/Z/16/Z) and Glasgow (104111/Z/14/Z).

PY - 2018/12/27

Y1 - 2018/12/27

N2 - Bloodstream form African trypanosomes are thought to rely exclusively upon glycolysis, using glucose as a substrate, for ATP production. Indeed, the pathway has long been considered a potential therapeutic target to tackle the devastating and neglected tropical diseases caused by these parasites. However, plasma membrane glucose and glycerol transporters are both expressed by trypanosomes and these parasites can infiltrate tissues that contain glycerol. Here, we show that bloodstream form trypanosomes can use glycerol for gluconeogenesis and for ATP production, particularly when deprived of glucose following hexose transporter depletion. We demonstrate that Trypanosoma brucei hexose transporters 1 and 2 (THT1 and THT2) are localized to the plasma membrane and that knockdown of THT1 expression leads to a growth defect that is more severe when THT2 is also knocked down. These data are consistent with THT1 and THT2 being the primary routes of glucose supply for the production of ATP by glycolysis. However, supplementation of the growth medium with glycerol substantially rescued the growth defect caused by THT1 and THT2 knockdown. Metabolomic analyses with heavy-isotope labelled glycerol demonstrated that trypanosomes take up glycerol and use it to synthesize intermediates of gluconeogenesis, including fructose 1,6-bisphosphate and hexose 6-phosphates, which feed the pentose phosphate pathway and variant surface glycoprotein biosynthesis. We used Cas9-mediated gene knockout to demonstrate a gluconeogenesis-specific, but fructose-1,6-bisphosphatase (Tb927.9.8720)-independent activity, converting fructose 1,6-bisphosphate into fructose 6-phosphate. In addition, we observed increased flux through the tricarboxylic acid cycle and the succinate shunt. Thus, contrary to prior thinking, gluconeogenesis can operate in bloodstream form T. brucei. This pathway, using glycerol as a physiological substrate, may be required in mammalian host tissues.

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