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Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites.

Pomel S, Luk FC, Beckers CJ - PLoS Pathog. (2008)

Bottom Line: Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress.Enzyme translocation is, however, not dependent on either F-actin or intact microtubules.We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, USA.

ABSTRACT
Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.

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Glycolysis is the primary source of energy for Toxoplasma motility.Toxoplasma tachyzoites were isolated from an infected monolayer 30–32 hours after infection as described in Materials and Methods. Parasites were collected by centrifugation and resuspended in EC buffer containing no carbon source or in the presence of glucose, glucose and KCN, or a mixture of pyruvate, lactate and glutamine. The effect of 10 nM atovaquone on Toxoplasma motility was analyzed in separate experiments. Incubations were performed in air or under a nitrogen atmosphere for 15 minutes at 37°C. Motility experiments were performed in quadruplicate and analyzed as described in Materials and Methods. The average fraction of motile parasites (±S.D.) under the different incubation conditions is shown.
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ppat-1000188-g001: Glycolysis is the primary source of energy for Toxoplasma motility.Toxoplasma tachyzoites were isolated from an infected monolayer 30–32 hours after infection as described in Materials and Methods. Parasites were collected by centrifugation and resuspended in EC buffer containing no carbon source or in the presence of glucose, glucose and KCN, or a mixture of pyruvate, lactate and glutamine. The effect of 10 nM atovaquone on Toxoplasma motility was analyzed in separate experiments. Incubations were performed in air or under a nitrogen atmosphere for 15 minutes at 37°C. Motility experiments were performed in quadruplicate and analyzed as described in Materials and Methods. The average fraction of motile parasites (±S.D.) under the different incubation conditions is shown.

Mentions: Toxoplasma invasion into and egress from its animal host cells are dependent on an actin-myosin-based motility system and thus require ATP for myosin action as well as actin filament formation. In most cells, the ATP necessary for these processes can be generated by glycolysis and by oxidative phosphorylation. The Toxoplasma genome encodes all the enzymes required for ATP production by glycolysis. It is not clear, however, whether apicomplexan parasites can produce ATP by oxidative phosphorylation given that pyruvate dehydrogenase, a key enzyme in coupling glycolysis and oxidative phosphorylation, appears to be absent from the parasite's mitochondria [7]–[11] as are some subunits of the ATP synthase [12],[13]. In order to better understand the roles of both metabolic pathways in supplying energy for parasite movement, we performed parasite motility assays under conditions where the parasites either had to rely on glycolysis for most of their energy production or on oxidative phosphorylation. Our observations are summarized in Figure 1. When assays were performed in the presence of glucose, a carbon source for both glycolysis and oxidative phosphorylation, we observed robust movement in a majority of the parasites (71.6±7.4%). Omission of glucose resulted in a drastic decrease in the fraction of mobile parasites (2.7±2.9%). When experiments were performed in the presence of glucose but under a nitrogen atmosphere or in the presence of KCN, conditions where any oxidative phosphorylation should be inhibited, Toxoplasma motility was not significantly affected with 70.5±12.4% and 63.1±16.1% of parasites moving, respectively. Although these observations indicate already that glycolysis is the major source of energy for parasite movement, they do not exclude the possibility that oxidative phosphorylation does make some contribution. To test this possibility, Toxoplasma motility experiments were performed in the absence of glucose but in the presence of potential substrates for the parasites' TCA cycle: pyruvate, lactate, or glutamine, both individually and in combination. As can be seen in Figure 1, only a small fraction of the parasites demonstrated motility (8.4±5.8%) and, as we observed in the absence of any substrate, this was limited to trails that were barely longer than the 2 µm cut-off limit (see Materials and Methods).


Host cell egress and invasion induce marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites.

Pomel S, Luk FC, Beckers CJ - PLoS Pathog. (2008)

Glycolysis is the primary source of energy for Toxoplasma motility.Toxoplasma tachyzoites were isolated from an infected monolayer 30–32 hours after infection as described in Materials and Methods. Parasites were collected by centrifugation and resuspended in EC buffer containing no carbon source or in the presence of glucose, glucose and KCN, or a mixture of pyruvate, lactate and glutamine. The effect of 10 nM atovaquone on Toxoplasma motility was analyzed in separate experiments. Incubations were performed in air or under a nitrogen atmosphere for 15 minutes at 37°C. Motility experiments were performed in quadruplicate and analyzed as described in Materials and Methods. The average fraction of motile parasites (±S.D.) under the different incubation conditions is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563030&req=5

ppat-1000188-g001: Glycolysis is the primary source of energy for Toxoplasma motility.Toxoplasma tachyzoites were isolated from an infected monolayer 30–32 hours after infection as described in Materials and Methods. Parasites were collected by centrifugation and resuspended in EC buffer containing no carbon source or in the presence of glucose, glucose and KCN, or a mixture of pyruvate, lactate and glutamine. The effect of 10 nM atovaquone on Toxoplasma motility was analyzed in separate experiments. Incubations were performed in air or under a nitrogen atmosphere for 15 minutes at 37°C. Motility experiments were performed in quadruplicate and analyzed as described in Materials and Methods. The average fraction of motile parasites (±S.D.) under the different incubation conditions is shown.
Mentions: Toxoplasma invasion into and egress from its animal host cells are dependent on an actin-myosin-based motility system and thus require ATP for myosin action as well as actin filament formation. In most cells, the ATP necessary for these processes can be generated by glycolysis and by oxidative phosphorylation. The Toxoplasma genome encodes all the enzymes required for ATP production by glycolysis. It is not clear, however, whether apicomplexan parasites can produce ATP by oxidative phosphorylation given that pyruvate dehydrogenase, a key enzyme in coupling glycolysis and oxidative phosphorylation, appears to be absent from the parasite's mitochondria [7]–[11] as are some subunits of the ATP synthase [12],[13]. In order to better understand the roles of both metabolic pathways in supplying energy for parasite movement, we performed parasite motility assays under conditions where the parasites either had to rely on glycolysis for most of their energy production or on oxidative phosphorylation. Our observations are summarized in Figure 1. When assays were performed in the presence of glucose, a carbon source for both glycolysis and oxidative phosphorylation, we observed robust movement in a majority of the parasites (71.6±7.4%). Omission of glucose resulted in a drastic decrease in the fraction of mobile parasites (2.7±2.9%). When experiments were performed in the presence of glucose but under a nitrogen atmosphere or in the presence of KCN, conditions where any oxidative phosphorylation should be inhibited, Toxoplasma motility was not significantly affected with 70.5±12.4% and 63.1±16.1% of parasites moving, respectively. Although these observations indicate already that glycolysis is the major source of energy for parasite movement, they do not exclude the possibility that oxidative phosphorylation does make some contribution. To test this possibility, Toxoplasma motility experiments were performed in the absence of glucose but in the presence of potential substrates for the parasites' TCA cycle: pyruvate, lactate, or glutamine, both individually and in combination. As can be seen in Figure 1, only a small fraction of the parasites demonstrated motility (8.4±5.8%) and, as we observed in the absence of any substrate, this was limited to trails that were barely longer than the 2 µm cut-off limit (see Materials and Methods).

Bottom Line: Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress.Enzyme translocation is, however, not dependent on either F-actin or intact microtubules.We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, USA.

ABSTRACT
Apicomplexan parasites are dependent on an F-actin and myosin-based motility system for their invasion into and escape from animal host cells, as well as for their general motility. In Toxoplasma gondii and Plasmodium species, the actin filaments and myosin motor required for this process are located in a narrow space between the parasite plasma membrane and the underlying inner membrane complex, a set of flattened cisternae that covers most the cytoplasmic face of the plasma membrane. Here we show that the energy required for Toxoplasma motility is derived mostly, if not entirely, from glycolysis and lactic acid production. We also demonstrate that the glycolytic enzymes of Toxoplasma tachyzoites undergo a striking relocation from the parasites' cytoplasm to their pellicles upon Toxoplasma egress from host cells. Specifically, it appears that the glycolytic enzymes are translocated to the cytoplasmic face of the inner membrane complex as well as to the space between the plasma membrane and inner membrane complex. The glycolytic enzymes remain pellicle-associated during extended incubations of parasites in the extracellular milieu and do not revert to a cytoplasmic location until well after parasites have completed invasion of new host cells. Translocation of glycolytic enzymes to and from the Toxoplasma pellicle appears to occur in response to changes in extracellular [K(+)] experienced during egress and invasion, a signal that requires changes of [Ca(2+)](c) in the parasite during egress. Enzyme translocation is, however, not dependent on either F-actin or intact microtubules. Our observations indicate that Toxoplasma gondii is capable of relocating its main source of energy between its cytoplasm and pellicle in response to exit from or entry into host cells. We propose that this ability allows Toxoplasma to optimize ATP delivery to those cellular processes that are most critical for survival outside host cells and those required for growth and replication of intracellular parasites.

Show MeSH
Related in: MedlinePlus