Limits...
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.

Show MeSH

Related in: MedlinePlus

Pellicle-association of aldolase-1 in extracellular Toxoplasma tachyzoites.(A) Extracellular parasites were disrupted at 4°C by sonication in 25 mM MOPS pH 7.0 containing 5 mM MgCl2 and 25 mM, 150 mM, or 300 mM KCl and subsequently fractionated into soluble and insoluble fraction by centrifugation. Equal parasite equivalents of the starting material, supernatant, and pellet fractions were separated by SDS-PAGE and analyzed by immunoblotting with monospecific antisera to Toxoplasma aldolase-1 and the membrane skeleton protein IMC1. (B) Extracellular parasites and intracellular parasites were harvested, homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl2 and 25 mM KCl as described above. The homogenate was fractionated into soluble and particulate material by centrifugation as above. The particulate fraction was subsequently extracted with 1% TX100 in the homogenization buffer and separated by centrifugation into detergent-soluble and insoluble fraction as described above. Equal parasite equivalents were analyzed by SDS-PAGe and immunoblotting with antisera to Toxoplasma aldolase-1, the membrane skeleton protein IMC1, the cytoplasmic protein CDPK1 and the plasma membrane protein SAG1. The starting material (TOT), supernatant (SN), and pellet (P) fractions were separated by SDS-PAGE and immuno-blotted with antisera to the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2563030&req=5

ppat-1000188-g009: Pellicle-association of aldolase-1 in extracellular Toxoplasma tachyzoites.(A) Extracellular parasites were disrupted at 4°C by sonication in 25 mM MOPS pH 7.0 containing 5 mM MgCl2 and 25 mM, 150 mM, or 300 mM KCl and subsequently fractionated into soluble and insoluble fraction by centrifugation. Equal parasite equivalents of the starting material, supernatant, and pellet fractions were separated by SDS-PAGE and analyzed by immunoblotting with monospecific antisera to Toxoplasma aldolase-1 and the membrane skeleton protein IMC1. (B) Extracellular parasites and intracellular parasites were harvested, homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl2 and 25 mM KCl as described above. The homogenate was fractionated into soluble and particulate material by centrifugation as above. The particulate fraction was subsequently extracted with 1% TX100 in the homogenization buffer and separated by centrifugation into detergent-soluble and insoluble fraction as described above. Equal parasite equivalents were analyzed by SDS-PAGe and immunoblotting with antisera to Toxoplasma aldolase-1, the membrane skeleton protein IMC1, the cytoplasmic protein CDPK1 and the plasma membrane protein SAG1. The starting material (TOT), supernatant (SN), and pellet (P) fractions were separated by SDS-PAGE and immuno-blotted with antisera to the indicated proteins.

Mentions: Association of aldolase-1 with the pellicle in extracellular parasites does not appear to involve a covalent or otherwise stable interaction with the IMC membrane or associated cytoskeletal elements. When extracellular parasites are mechanically disrupted at approximate physiological salt concentrations (150 mM) and subjected to differential centrifugation, the majority of aldolase-1 is recovered in the supernatant. We noticed, however, that 20–30% of aldolase-1 was consistently recovered in the pellet fraction (Figure 9A). This association was sensitive to the salt concentration in the extraction buffer. When extractions were performed at higher KCl concentration (300 mM) all aldolase was recovered in the soluble fraction. At 25 mM KCl, on the other hand, we recovered 70–80% of aldolase-1 in the pellet fraction. To analyze this phenomenon in greater detail we compared the fractionation of aldolase-1 in intracellular and extracellular Toxoplasma at 25 mM KCl. As can be seen in Figure 9B, pellicle association of aldolase-1 is only observed in extracellular parasites under these conditions. The association is specific in that the cytoplasmic enzyme CDPK1 is completely solubilized under these conditions. Pellicle-association of aldolase-1 appears to require intact membranes as addition of Triton –X100 result in the complete solubilization of aldolase-1.


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

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

Pellicle-association of aldolase-1 in extracellular Toxoplasma tachyzoites.(A) Extracellular parasites were disrupted at 4°C by sonication in 25 mM MOPS pH 7.0 containing 5 mM MgCl2 and 25 mM, 150 mM, or 300 mM KCl and subsequently fractionated into soluble and insoluble fraction by centrifugation. Equal parasite equivalents of the starting material, supernatant, and pellet fractions were separated by SDS-PAGE and analyzed by immunoblotting with monospecific antisera to Toxoplasma aldolase-1 and the membrane skeleton protein IMC1. (B) Extracellular parasites and intracellular parasites were harvested, homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl2 and 25 mM KCl as described above. The homogenate was fractionated into soluble and particulate material by centrifugation as above. The particulate fraction was subsequently extracted with 1% TX100 in the homogenization buffer and separated by centrifugation into detergent-soluble and insoluble fraction as described above. Equal parasite equivalents were analyzed by SDS-PAGe and immunoblotting with antisera to Toxoplasma aldolase-1, the membrane skeleton protein IMC1, the cytoplasmic protein CDPK1 and the plasma membrane protein SAG1. The starting material (TOT), supernatant (SN), and pellet (P) fractions were separated by SDS-PAGE and immuno-blotted with antisera to the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000188-g009: Pellicle-association of aldolase-1 in extracellular Toxoplasma tachyzoites.(A) Extracellular parasites were disrupted at 4°C by sonication in 25 mM MOPS pH 7.0 containing 5 mM MgCl2 and 25 mM, 150 mM, or 300 mM KCl and subsequently fractionated into soluble and insoluble fraction by centrifugation. Equal parasite equivalents of the starting material, supernatant, and pellet fractions were separated by SDS-PAGE and analyzed by immunoblotting with monospecific antisera to Toxoplasma aldolase-1 and the membrane skeleton protein IMC1. (B) Extracellular parasites and intracellular parasites were harvested, homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl2 and 25 mM KCl as described above. The homogenate was fractionated into soluble and particulate material by centrifugation as above. The particulate fraction was subsequently extracted with 1% TX100 in the homogenization buffer and separated by centrifugation into detergent-soluble and insoluble fraction as described above. Equal parasite equivalents were analyzed by SDS-PAGe and immunoblotting with antisera to Toxoplasma aldolase-1, the membrane skeleton protein IMC1, the cytoplasmic protein CDPK1 and the plasma membrane protein SAG1. The starting material (TOT), supernatant (SN), and pellet (P) fractions were separated by SDS-PAGE and immuno-blotted with antisera to the indicated proteins.
Mentions: Association of aldolase-1 with the pellicle in extracellular parasites does not appear to involve a covalent or otherwise stable interaction with the IMC membrane or associated cytoskeletal elements. When extracellular parasites are mechanically disrupted at approximate physiological salt concentrations (150 mM) and subjected to differential centrifugation, the majority of aldolase-1 is recovered in the supernatant. We noticed, however, that 20–30% of aldolase-1 was consistently recovered in the pellet fraction (Figure 9A). This association was sensitive to the salt concentration in the extraction buffer. When extractions were performed at higher KCl concentration (300 mM) all aldolase was recovered in the soluble fraction. At 25 mM KCl, on the other hand, we recovered 70–80% of aldolase-1 in the pellet fraction. To analyze this phenomenon in greater detail we compared the fractionation of aldolase-1 in intracellular and extracellular Toxoplasma at 25 mM KCl. As can be seen in Figure 9B, pellicle association of aldolase-1 is only observed in extracellular parasites under these conditions. The association is specific in that the cytoplasmic enzyme CDPK1 is completely solubilized under these conditions. Pellicle-association of aldolase-1 appears to require intact membranes as addition of Triton –X100 result in the complete solubilization of aldolase-1.

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