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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: 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.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.

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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Aldolase-1 is associated with the cytoplasmic face of the IMC in extracellular Toxoplasma.Toxoplasma aldolase-1 is associated with the cytoplasmic face of the IMC. Intracellular (A, B) and extracellular (C–F) Toxoplasma tachyzoites were homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl2 containing either 25 mM (A, C, D, F) or 300 mM KCl (B, E) and fractionated as described in Fig. 8A. Isolated pellicle fractions were incubated with antiserum to Toxoplasma aldolase-1 (A–E) or non-specific antiserum (F) and gold-conjugated secondary antibodies in the same extraction buffer and subsequently processed as described in Materials and Methods. Bars = 200 nm. Asterisks indicate the subpellicular microtubules on the cytoplasmic face of the IMC.
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ppat-1000188-g010: Aldolase-1 is associated with the cytoplasmic face of the IMC in extracellular Toxoplasma.Toxoplasma aldolase-1 is associated with the cytoplasmic face of the IMC. Intracellular (A, B) and extracellular (C–F) Toxoplasma tachyzoites were homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl2 containing either 25 mM (A, C, D, F) or 300 mM KCl (B, E) and fractionated as described in Fig. 8A. Isolated pellicle fractions were incubated with antiserum to Toxoplasma aldolase-1 (A–E) or non-specific antiserum (F) and gold-conjugated secondary antibodies in the same extraction buffer and subsequently processed as described in Materials and Methods. Bars = 200 nm. Asterisks indicate the subpellicular microtubules on the cytoplasmic face of the IMC.

Mentions: To determine if the salt-resistant fraction of aldolase-1 in extracellular Toxoplasma is indeed IMC associated, we homogenized parasites in 25 mM or 300 mM KCl buffer and performed pre-embedding immuno-electron microscopy on the isolated membrane fractions. It should be noted that association of the plasma membrane with the outer face of the IMC did not appear to be affected at the KCl concentrations used in this study. As can be seen in Figure 10 (panels A, B), no aldolase was detected in association with the pellicle of intracellular Toxoplasma prepared in 25 mM KCl. In pellicles isolated from extracellular parasites at 25 mM KCl labeling of the cytoplasmic face of the IMC was readily detected (Figure 10, panels C and D). This labeling was absent from pellicles in samples prepared in the presence of 300 mM KCl (Figure 10, panel E). Taken together with the data shown in Figures 3, 4, and 5 these findings indicate that a substantial fraction of aldolase-1 in extracellular Toxoplasma is intimately associated with the cytoplasmic face of the IMC membrane.


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

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

Aldolase-1 is associated with the cytoplasmic face of the IMC in extracellular Toxoplasma.Toxoplasma aldolase-1 is associated with the cytoplasmic face of the IMC. Intracellular (A, B) and extracellular (C–F) Toxoplasma tachyzoites were homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl2 containing either 25 mM (A, C, D, F) or 300 mM KCl (B, E) and fractionated as described in Fig. 8A. Isolated pellicle fractions were incubated with antiserum to Toxoplasma aldolase-1 (A–E) or non-specific antiserum (F) and gold-conjugated secondary antibodies in the same extraction buffer and subsequently processed as described in Materials and Methods. Bars = 200 nm. Asterisks indicate the subpellicular microtubules on the cytoplasmic face of the IMC.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000188-g010: Aldolase-1 is associated with the cytoplasmic face of the IMC in extracellular Toxoplasma.Toxoplasma aldolase-1 is associated with the cytoplasmic face of the IMC. Intracellular (A, B) and extracellular (C–F) Toxoplasma tachyzoites were homogenized in 25 mM MOPS pH 7.0, 5 mM MgCl2 containing either 25 mM (A, C, D, F) or 300 mM KCl (B, E) and fractionated as described in Fig. 8A. Isolated pellicle fractions were incubated with antiserum to Toxoplasma aldolase-1 (A–E) or non-specific antiserum (F) and gold-conjugated secondary antibodies in the same extraction buffer and subsequently processed as described in Materials and Methods. Bars = 200 nm. Asterisks indicate the subpellicular microtubules on the cytoplasmic face of the IMC.
Mentions: To determine if the salt-resistant fraction of aldolase-1 in extracellular Toxoplasma is indeed IMC associated, we homogenized parasites in 25 mM or 300 mM KCl buffer and performed pre-embedding immuno-electron microscopy on the isolated membrane fractions. It should be noted that association of the plasma membrane with the outer face of the IMC did not appear to be affected at the KCl concentrations used in this study. As can be seen in Figure 10 (panels A, B), no aldolase was detected in association with the pellicle of intracellular Toxoplasma prepared in 25 mM KCl. In pellicles isolated from extracellular parasites at 25 mM KCl labeling of the cytoplasmic face of the IMC was readily detected (Figure 10, panels C and D). This labeling was absent from pellicles in samples prepared in the presence of 300 mM KCl (Figure 10, panel E). Taken together with the data shown in Figures 3, 4, and 5 these findings indicate that a substantial fraction of aldolase-1 in extracellular Toxoplasma is intimately associated with the cytoplasmic face of the IMC membrane.

Bottom Line: 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.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.

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