Glutamine Utilization and GABA Shunt

Intracellular and extracellular Toxoplasma parasites can utilize glutamine in addition to glucose as source of carbon under normal growth conditions. It has been thought for a long time that T. gondii relies on glucose as the only carbon source from host for its metabolism. It has been recently found neither the host glucose nor is the glucose transporter essential to the growth of parasites in vitro. The genetic disruption of the parasite glucose uptake in the intracellular parasites had no effect on growth and the egressed parasites lacking glucose transporters showed restored gliding motility when glutamine was supplemented in the media [1]. This suggests that T. gondii tachyzoites can utilise glutamine in response to glucose starvation.

 

The presence of PDH complex in the apicoplast of T. gondii and Neospora caninum and many other apicomplexans as opposed its mitochondrial location in other organisms suggests the disconnection of glycolysis from TCA cycle. Studies have suggested that both the isoforms of NADH dehydrogenase are essential for the growth of T. gondii and even single knockout mutants displayed a decreased replication rate and also decreased mitochondrial membrane potential [2]. The mitochondrial respiratory chain has also been proved to be essential in Plasmodium yoeli as the inhibition of electron transport and mitochondrial depolarization with atovaquone treatment caused cellular damage and death [3]. The same effect was observed with atovaquone in T. gondii [4]. All these evidence suggest that the respiratory chain and oxidative phosphorylation are functional in apicomplexans T. gondii and Plasmodium species, although the pathway differs in several aspects from that of the hosts [5]. Although the functional respiratory chain is essential for parasite survival, the conditional knockout of the TCA cycle enzyme, succinyl-CoA synthetase resulted only in 30% reduction in growth rate and no alteration in the mitochondrial membrane potential was observed. This suggests that functional TCA cycle is not essential for parasite replication.

 

Metabolic profiling studies by MacRae et al showed that glucose is catabolised through a complete functional TCA cycle in intracellular and egressed tachyzoites. The inhibition of the TCA cycle enzyme citrate synthase also resulted in the partial inhibition of apicoplast-based de novo fatty acid biosynthesis pathway. Fatty acid biosynthesis pathway requires the reducing equivalent NADPH and it was suggested that TCA cycle enzymes replenish NADPH [6]. Immuno-localization studies showed that T. gondii aconitase enzyme is dually targeted to mitochondria and apicoplast and one of the two isoforms of isocitrate dehydrogenase (ICDH1) exhibits apicoplast localization [7]. This shows that citrate is not only a substrate for mitochondria-based TCA cycle and energy production and a partial TCA pathway also exists in the apicoplast. Therefore, aconitase and isocitrate dehydrogenase will catalyse the production of 2-oxoglutarate in the apicoplast, also leading to regeneration of NADPH. There were no experimental studies carried out to localise one of the two isoforms of citrate synthase, while the other isoform is localised to mitochondria [8]. Therefore, it is not clear whether citrate produced in the mitochondria is transported to apicoplast, where the remainder of the pathway takes place or the citrate is produced de novo in the apicoplast. This pathway has been drawn with available evidence and therefore it is drawn on the assumption that mitochondrial citrate is transported to the apicoplast.

 

The T. gondii, P. falciparum and N. caninum genomes possess genes putatively annotated as lysine decarboxylase previously. These proteins contain pyridoxal phosphate-dependent amino acid decarboxylase domains which share conserved domains with members of the bacterial amino acid decarboxylase superfamily [9]. The product of lysine decarboxylase enzyme, cadaverine was not detectable in the metabolic profiling studies in T. gondii. Experimental studies carried out with the null mutant of this gene in T. gondii had undetectable levels of GABA and elevated levels of glutamate. This was also accompanied by changes in the levels of other metabolites involved in central carbon metabolism. The lack of glutamine decarboxylase (GAD) activity in this null mutant suggests that this is the only gene in T. gondii genome that encodes GAD. In addition, the GAD activity was only observed even in wild type parasites when lysates were supplemented with ATP. This suggests that the activation of this enzyme requires ATP. MacRae et al also suggests the presence of genes for other genes of GABA shunt. These include glutamate transamidase, putatively annotated as ornithine transamidase, succinic-semialdehyde dehydrogenase and a GABA transporter, electronically annotated to contain sodium:neurotransmitter transporter domain. Homologs of these genes were also identified in N. caninum [7].

 

Enzyme EC Number Gene ID
Isocitrate dehydrogenase (ICDH1) 1.1.1.42 NCLIV_039280
Succinate semialdehyde dehydrogenase 1.2.1.16 NCLIV_068990
Glutamate dehydrogenase 1.4.1.4 NCLIV_000130
Glutamate transaminase 2.6.1.19 NCLIV_037280
Glutamate decarboxylase 4.1.1.15 NCLIV_019490
Aconitate hydratase 4.2.1.3 NCLIV_046260
Glutamate-ammonia ligase 6.3.1.2 NCLIV_034160
GABA transporter none NCLIV_003090
GABA transporter none NCLIV_014580
Oxoglutarate/malate translocator none NCLIV_033680
Amino acid transporter none NCLIV_043960

 

 

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Sources and fates of metabolites

 

Substrate Source pathways Product Fate pathways
Glutamine Host GABA Host
Citrate Tricarboxylic acid (TCA) cycle Succinate Tricarboxylic acid (TCA) cycle
NADP(+) Fatty acid biosynthesis in the apicoplast NADPH Fatty acid biosynthesis in the apicoplast
Glutamate Glutamate metabolism 2-oxoglutarate Tricarboxylic acid (TCA) cycle
2-oxoglutarate Tricarboxylic acid (TCA) cycle