Glutamine Utilization and GABA Shunt

Intracellular and extracellular 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 uptake and catabolism of glutamine has also been observed in the malaria parasites, Plasmodium species. The TCA cycle plays a minor role in the asexual blood-stages of Plasmodium falciparum, although the genome encodes all essential genes for functional TCA cycle. It is also known that the TCA cycle is disconnected from glycolysis, especially with the presence of pyruvate dehydrogenase (PDH) complex in apicoplast. It has been identified by Olszewski et al that TCA metabolism is not a cyclical pathway at least in the intraerythrocytic stages and it forms a branched pathway. It was evident from metabolic profiling that glutamine was used as a carbon source by Plasmodium and 2-oxoglutarate, an intermediate of TCA metabolism was generated from it. This bifurcated pathway is essential to the mitochondrial generation of acetyl-CoA as opposed to the conventional PDH complex apicoplast. This mitochondrial acetyl-CoA is utilized in the histone acetylation opposed to glucose-derived acetyl-CoA for amino sugar acetylation [2].

 

The presence of PDH complex in apicoplast in T. gondii 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 [3]. 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 [4]. The same effect was observed with atovaquone in T. gondii [5]. 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 [6]. 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 [7]. 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 [8]. 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 [9]. 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 Neospora 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 [10]. The product of lysine decarboxylase enzyme, cadaverine was not detectable in the metabolic profiling studies. 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 life 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 the closest Coccidian N. caninum as well as P. falciparum. The experimental results also showed that although GABA shunt is important in parasite fitness, it is not essential for survival of parasites. It was also evident that GABA shunt is required to sustain normal motility in nutrient-deprived conditions. In addition, the tachyzoites stages utilize GABA and is not actively secreted opposed to release of GABA in bradyzoite stages [7]. The secretion of GABA in bradyzoite may also be responsible for behavioural changes in T. gondii infected individuals [11,12,13].

 

Enzyme EC Number Gene ID Protein localisation Localisation data source
Isocitrate dehydrogenase (ICDH1) 1.1.1.42 TGME49_266760 Apicoplast Apiloc
Succinate semialdehyde dehydrogenase 1.2.1.16 TGME49_257480 Mitochondria Prediction
Glutamate dehydrogenase 1.4.1.4 TGME49_293180 Cytosol Prediction
Glutamate transaminase 2.6.1.19 TGME49_269110    
Glutamate decarboxylase 4.1.1.15 TGME49_280700 Mitochondria Prediction
Aconitate hydratase 4.2.1.3 TGME49_226730 Mitochondria, Apicoplast Apiloc
Glutamate-ammonia ligase 6.3.1.2 TGME49_273490 Mitochondria Prediction
GABA transporter none TGME49_208420 Motochondrial membrane  
Oxoglutarate/malate transporter none TGME49_274060 Mitochondrial inner membrane Apiloc, GO annotation
GABA transporter none TGME49_285800    
Amino acid transporter none TGME49_305470    

 

 

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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