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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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Related in: MedlinePlus

Aldolase-1 relocation during egress requires a decrease in environmental [K+] and an increase in [Ca2+]c and does not require either F-actin or microtubules.(A) Intracellular parasites expressing myc-aldolase-1 were harvested 24 hours after infection in IC buffer as described in Materials and Methods and subsequently incubated for the indicated times in IC buffer at 37°C. After 60 minutes in IC buffer, parasites were recovered by centrifugation, resuspended in EC buffer, and incubated at 37°C for the indicated time. The fraction of parasites with peripheral aldolase-1 was determined as described above. (B) Intracellular parasites expressing myc-aldolase-1 were harvested 24 hours after infection in EC buffer as described in Materials and Methods and subsequently incubated for the indicated times in EC buffer at 37°C before fixation and processing for immunofluorescence microscopy as in Figure 4. The fraction of parasites with peripheral aldolase-1 was determined as in panel A. (C) Intracellular parasites expressing myc-tagged aldolase-1 were released from host cells in IC buffer and subsequently switched to EC buffer. Extracellular parasites expressing myc-tagged aldolase-1 were resuspended in EC buffer and further switched to IC buffer. Each buffer was supplemented by DMSO (control) or BAPTA-AM (20 µM). Parasites were incubated in each buffer for 30 min at 37°C and processed for immunofluorescence using mouse anti-myc antibody (green) and rabbit anti-IMC1 antiserum (red). Bars = 2 µm. (D) Intracellular parasites expressing myc-tagged aldolase-1 were treated with DMSO or 10 µM cytochalasin D (CytD) for 15 min at 37°C and subsequently released from host cells by passage through a 25G needle. Motility assays were performed in the presence of DMSO or cytochalasin D as described in Materials and Methods. Parasites were labeled using anti-myc (mALD1, green) and anti-SAG1 (red) antibodies. In the top panel, note the presence of a motile (top) and a non-motile (bottom) parasite. No motility was detected after cytochalasin D treatment. Overlay pictures of DIC and DAPI are shown on the right. Bars = 2 µm. (E) Intracellular parasites were treated for 24 hours with 2.5 µM oryzalin and subsequently released from host cells by passage through a 25G needle into EC buffer. After 15 minutes at 37°C, parasites were fixed and processed for immunofluorescence microscopy using anti-myc (mALD1, green) and anti-IMC1 (red) antiserum. An overlay of the DIC and DAPI are shown on the right. All samples were fixed in −20°C methanol. Bar = 2 µm.
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ppat-1000188-g007: Aldolase-1 relocation during egress requires a decrease in environmental [K+] and an increase in [Ca2+]c and does not require either F-actin or microtubules.(A) Intracellular parasites expressing myc-aldolase-1 were harvested 24 hours after infection in IC buffer as described in Materials and Methods and subsequently incubated for the indicated times in IC buffer at 37°C. After 60 minutes in IC buffer, parasites were recovered by centrifugation, resuspended in EC buffer, and incubated at 37°C for the indicated time. The fraction of parasites with peripheral aldolase-1 was determined as described above. (B) Intracellular parasites expressing myc-aldolase-1 were harvested 24 hours after infection in EC buffer as described in Materials and Methods and subsequently incubated for the indicated times in EC buffer at 37°C before fixation and processing for immunofluorescence microscopy as in Figure 4. The fraction of parasites with peripheral aldolase-1 was determined as in panel A. (C) Intracellular parasites expressing myc-tagged aldolase-1 were released from host cells in IC buffer and subsequently switched to EC buffer. Extracellular parasites expressing myc-tagged aldolase-1 were resuspended in EC buffer and further switched to IC buffer. Each buffer was supplemented by DMSO (control) or BAPTA-AM (20 µM). Parasites were incubated in each buffer for 30 min at 37°C and processed for immunofluorescence using mouse anti-myc antibody (green) and rabbit anti-IMC1 antiserum (red). Bars = 2 µm. (D) Intracellular parasites expressing myc-tagged aldolase-1 were treated with DMSO or 10 µM cytochalasin D (CytD) for 15 min at 37°C and subsequently released from host cells by passage through a 25G needle. Motility assays were performed in the presence of DMSO or cytochalasin D as described in Materials and Methods. Parasites were labeled using anti-myc (mALD1, green) and anti-SAG1 (red) antibodies. In the top panel, note the presence of a motile (top) and a non-motile (bottom) parasite. No motility was detected after cytochalasin D treatment. Overlay pictures of DIC and DAPI are shown on the right. Bars = 2 µm. (E) Intracellular parasites were treated for 24 hours with 2.5 µM oryzalin and subsequently released from host cells by passage through a 25G needle into EC buffer. After 15 minutes at 37°C, parasites were fixed and processed for immunofluorescence microscopy using anti-myc (mALD1, green) and anti-IMC1 (red) antiserum. An overlay of the DIC and DAPI are shown on the right. All samples were fixed in −20°C methanol. Bar = 2 µm.

Mentions: It appears that translocation of aldolase-1 from the cytoplasm to the pellicle only occurs at or shortly after Toxoplasma egress from its host cells. Egress is not synchronous or predictable under physiological conditions but is readily induced by the physical disruption of the host cells [20]. Although earlier time points could not be obtained for practical reasons, aldolase-1 translocation to the Toxoplasma pellicle could be detected as early as 30 seconds after parasite egress and was essentially complete by 60 minutes (Figure 7A).


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 relocation during egress requires a decrease in environmental [K+] and an increase in [Ca2+]c and does not require either F-actin or microtubules.(A) Intracellular parasites expressing myc-aldolase-1 were harvested 24 hours after infection in IC buffer as described in Materials and Methods and subsequently incubated for the indicated times in IC buffer at 37°C. After 60 minutes in IC buffer, parasites were recovered by centrifugation, resuspended in EC buffer, and incubated at 37°C for the indicated time. The fraction of parasites with peripheral aldolase-1 was determined as described above. (B) Intracellular parasites expressing myc-aldolase-1 were harvested 24 hours after infection in EC buffer as described in Materials and Methods and subsequently incubated for the indicated times in EC buffer at 37°C before fixation and processing for immunofluorescence microscopy as in Figure 4. The fraction of parasites with peripheral aldolase-1 was determined as in panel A. (C) Intracellular parasites expressing myc-tagged aldolase-1 were released from host cells in IC buffer and subsequently switched to EC buffer. Extracellular parasites expressing myc-tagged aldolase-1 were resuspended in EC buffer and further switched to IC buffer. Each buffer was supplemented by DMSO (control) or BAPTA-AM (20 µM). Parasites were incubated in each buffer for 30 min at 37°C and processed for immunofluorescence using mouse anti-myc antibody (green) and rabbit anti-IMC1 antiserum (red). Bars = 2 µm. (D) Intracellular parasites expressing myc-tagged aldolase-1 were treated with DMSO or 10 µM cytochalasin D (CytD) for 15 min at 37°C and subsequently released from host cells by passage through a 25G needle. Motility assays were performed in the presence of DMSO or cytochalasin D as described in Materials and Methods. Parasites were labeled using anti-myc (mALD1, green) and anti-SAG1 (red) antibodies. In the top panel, note the presence of a motile (top) and a non-motile (bottom) parasite. No motility was detected after cytochalasin D treatment. Overlay pictures of DIC and DAPI are shown on the right. Bars = 2 µm. (E) Intracellular parasites were treated for 24 hours with 2.5 µM oryzalin and subsequently released from host cells by passage through a 25G needle into EC buffer. After 15 minutes at 37°C, parasites were fixed and processed for immunofluorescence microscopy using anti-myc (mALD1, green) and anti-IMC1 (red) antiserum. An overlay of the DIC and DAPI are shown on the right. All samples were fixed in −20°C methanol. Bar = 2 µm.
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Related In: Results  -  Collection

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ppat-1000188-g007: Aldolase-1 relocation during egress requires a decrease in environmental [K+] and an increase in [Ca2+]c and does not require either F-actin or microtubules.(A) Intracellular parasites expressing myc-aldolase-1 were harvested 24 hours after infection in IC buffer as described in Materials and Methods and subsequently incubated for the indicated times in IC buffer at 37°C. After 60 minutes in IC buffer, parasites were recovered by centrifugation, resuspended in EC buffer, and incubated at 37°C for the indicated time. The fraction of parasites with peripheral aldolase-1 was determined as described above. (B) Intracellular parasites expressing myc-aldolase-1 were harvested 24 hours after infection in EC buffer as described in Materials and Methods and subsequently incubated for the indicated times in EC buffer at 37°C before fixation and processing for immunofluorescence microscopy as in Figure 4. The fraction of parasites with peripheral aldolase-1 was determined as in panel A. (C) Intracellular parasites expressing myc-tagged aldolase-1 were released from host cells in IC buffer and subsequently switched to EC buffer. Extracellular parasites expressing myc-tagged aldolase-1 were resuspended in EC buffer and further switched to IC buffer. Each buffer was supplemented by DMSO (control) or BAPTA-AM (20 µM). Parasites were incubated in each buffer for 30 min at 37°C and processed for immunofluorescence using mouse anti-myc antibody (green) and rabbit anti-IMC1 antiserum (red). Bars = 2 µm. (D) Intracellular parasites expressing myc-tagged aldolase-1 were treated with DMSO or 10 µM cytochalasin D (CytD) for 15 min at 37°C and subsequently released from host cells by passage through a 25G needle. Motility assays were performed in the presence of DMSO or cytochalasin D as described in Materials and Methods. Parasites were labeled using anti-myc (mALD1, green) and anti-SAG1 (red) antibodies. In the top panel, note the presence of a motile (top) and a non-motile (bottom) parasite. No motility was detected after cytochalasin D treatment. Overlay pictures of DIC and DAPI are shown on the right. Bars = 2 µm. (E) Intracellular parasites were treated for 24 hours with 2.5 µM oryzalin and subsequently released from host cells by passage through a 25G needle into EC buffer. After 15 minutes at 37°C, parasites were fixed and processed for immunofluorescence microscopy using anti-myc (mALD1, green) and anti-IMC1 (red) antiserum. An overlay of the DIC and DAPI are shown on the right. All samples were fixed in −20°C methanol. Bar = 2 µm.
Mentions: It appears that translocation of aldolase-1 from the cytoplasm to the pellicle only occurs at or shortly after Toxoplasma egress from its host cells. Egress is not synchronous or predictable under physiological conditions but is readily induced by the physical disruption of the host cells [20]. Although earlier time points could not be obtained for practical reasons, aldolase-1 translocation to the Toxoplasma pellicle could be detected as early as 30 seconds after parasite egress and was essentially complete by 60 minutes (Figure 7A).

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