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Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth.

Kurokawa H, Kato K, Iwanaga T, Sugi T, Sudo A, Kobayashi K, Gong H, Takemae H, Recuenco FC, Horimoto T, Akashi H - PLoS ONE (2011)

Bottom Line: The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated.Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT

Background: cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/principal finding: The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/significance: Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

Show MeSH
T. gondii tachyzoite growth assay.Average parasite number per parasitophorous vacuole at 12, 24, and 36 h postinfection. RH/PKA-R or RH/GFP parasites were added to Vero cell monolayers at a MOI of 1∶10 and incubated for 2 h at 37°C. Extracellular parasites were then removed by washing three times with PBS(–) and returned to culture in complete medium with or without PKA inhibitor. The final concentration of inhibitors H89 (Promega) and PKI14-22 (Calbiochem) was 2 µM. P/V: parasites per vacuole. Representative results of three independent determinations are shown.
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pone-0022492-g005: T. gondii tachyzoite growth assay.Average parasite number per parasitophorous vacuole at 12, 24, and 36 h postinfection. RH/PKA-R or RH/GFP parasites were added to Vero cell monolayers at a MOI of 1∶10 and incubated for 2 h at 37°C. Extracellular parasites were then removed by washing three times with PBS(–) and returned to culture in complete medium with or without PKA inhibitor. The final concentration of inhibitors H89 (Promega) and PKI14-22 (Calbiochem) was 2 µM. P/V: parasites per vacuole. Representative results of three independent determinations are shown.

Mentions: To investigate the role of TgPKA in the asexual stage, we performed a tachyzoite growth assay in the presence of H89 and PKI. Tachyzoites treated with H89 have been reported to show an increased doubling time [19], indicating a potential role of TgPKA in replication. However, an effect of H89 on host cell PKA still cannot be ruled out, since mammalian PKA showed high susceptibility to H89 in the in vitro kinase assay (Figure 4B). To clarify whether the growth defect was due to inhibition of T. gondii or host cell PKA, we compared the effect of H89 with that of PKI in a tachyzoite growth assay. Since PKI5–24, which we used in the in vitro kinase assay, is not cell-permeable, we used the cell-permeable, myristolated PKI14–22. The final concentration of both H89 [19] and PKI was 2 µM. Figure 5 shows the average parasite number per parasitophorous vacuole (P/V) at 12, 24, and 36 h post-invasion. Vacuoles containing more than 32 parasites were calculated as 32 P/V, since above this value discriminating individual parasites became problematic. The average P/V in controls was 1.71, 6.22, and 28.3 at each time point. In dimethyl sulfoxide (DMSO)-treated parasites (a vehicle of H89), the average P/V was 1.64, 6.90, and 27.7 at each time point, similar to those of the control parasites. The average P/V of H89-treated parasites was markedly lower (1.63, 4.61, and 10.7). The growth rate of H89-treated parasites at 36 h was about half that of the control, suggesting growth inhibition. This is consistent with previous reports [19]. In contrast, in mammalian PKI-treated parasites, the average P/V values were 1.62, 5.35, and 29.4. Since no significant difference between the P/V of the control and PKI-treated parasites was observed, PKI seems to have no inhibitory effect on tachyzoite growth. This result indicates that the susceptibility of TgPKA-C to PKI is lower than that to H89.


Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth.

Kurokawa H, Kato K, Iwanaga T, Sugi T, Sudo A, Kobayashi K, Gong H, Takemae H, Recuenco FC, Horimoto T, Akashi H - PLoS ONE (2011)

T. gondii tachyzoite growth assay.Average parasite number per parasitophorous vacuole at 12, 24, and 36 h postinfection. RH/PKA-R or RH/GFP parasites were added to Vero cell monolayers at a MOI of 1∶10 and incubated for 2 h at 37°C. Extracellular parasites were then removed by washing three times with PBS(–) and returned to culture in complete medium with or without PKA inhibitor. The final concentration of inhibitors H89 (Promega) and PKI14-22 (Calbiochem) was 2 µM. P/V: parasites per vacuole. Representative results of three independent determinations are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140512&req=5

pone-0022492-g005: T. gondii tachyzoite growth assay.Average parasite number per parasitophorous vacuole at 12, 24, and 36 h postinfection. RH/PKA-R or RH/GFP parasites were added to Vero cell monolayers at a MOI of 1∶10 and incubated for 2 h at 37°C. Extracellular parasites were then removed by washing three times with PBS(–) and returned to culture in complete medium with or without PKA inhibitor. The final concentration of inhibitors H89 (Promega) and PKI14-22 (Calbiochem) was 2 µM. P/V: parasites per vacuole. Representative results of three independent determinations are shown.
Mentions: To investigate the role of TgPKA in the asexual stage, we performed a tachyzoite growth assay in the presence of H89 and PKI. Tachyzoites treated with H89 have been reported to show an increased doubling time [19], indicating a potential role of TgPKA in replication. However, an effect of H89 on host cell PKA still cannot be ruled out, since mammalian PKA showed high susceptibility to H89 in the in vitro kinase assay (Figure 4B). To clarify whether the growth defect was due to inhibition of T. gondii or host cell PKA, we compared the effect of H89 with that of PKI in a tachyzoite growth assay. Since PKI5–24, which we used in the in vitro kinase assay, is not cell-permeable, we used the cell-permeable, myristolated PKI14–22. The final concentration of both H89 [19] and PKI was 2 µM. Figure 5 shows the average parasite number per parasitophorous vacuole (P/V) at 12, 24, and 36 h post-invasion. Vacuoles containing more than 32 parasites were calculated as 32 P/V, since above this value discriminating individual parasites became problematic. The average P/V in controls was 1.71, 6.22, and 28.3 at each time point. In dimethyl sulfoxide (DMSO)-treated parasites (a vehicle of H89), the average P/V was 1.64, 6.90, and 27.7 at each time point, similar to those of the control parasites. The average P/V of H89-treated parasites was markedly lower (1.63, 4.61, and 10.7). The growth rate of H89-treated parasites at 36 h was about half that of the control, suggesting growth inhibition. This is consistent with previous reports [19]. In contrast, in mammalian PKI-treated parasites, the average P/V values were 1.62, 5.35, and 29.4. Since no significant difference between the P/V of the control and PKI-treated parasites was observed, PKI seems to have no inhibitory effect on tachyzoite growth. This result indicates that the susceptibility of TgPKA-C to PKI is lower than that to H89.

Bottom Line: The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated.Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT

Background: cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/principal finding: The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/significance: Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

Show MeSH