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A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3.

Yamamoto M, Standley DM, Takashima S, Saiga H, Okuyama M, Kayama H, Kubo E, Ito H, Takaura M, Matsuda T, Soldati-Favre D, Takeda K - J. Exp. Med. (2009)

Bottom Line: We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages.In addition, kinase-inactive ROP16 failed to activate Stat3.These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT
Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

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The L503S polymorphic mutation in type II ROP16 determines its defective Stat3 activation. (A–D) 293T cells were transfected with the APRE-luc plasmid together with the indicated ROP16 expression vectors. Luciferase activities were expressed as fold increases over the background levels shown by lysates prepared from mock-transfected cells. Indicated values are means ± the variation range of duplicates. A list of the chimeric ROP16 expression vectors is shown (B, left). R, RH (pink); M, ME49 (blue). (E) Serum-starved MEFs were infected with an MOI = 10 of the indicated parasites for 18 h. Activation of Stat3 was also determined by Western blot analysis of cell extracts using anti–phospho-Stat3 Tyr 705. Stat3 and actin levels are shown as loading controls. Data are representative of three (A and E) or two (B–D) independent experiments.
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fig4: The L503S polymorphic mutation in type II ROP16 determines its defective Stat3 activation. (A–D) 293T cells were transfected with the APRE-luc plasmid together with the indicated ROP16 expression vectors. Luciferase activities were expressed as fold increases over the background levels shown by lysates prepared from mock-transfected cells. Indicated values are means ± the variation range of duplicates. A list of the chimeric ROP16 expression vectors is shown (B, left). R, RH (pink); M, ME49 (blue). (E) Serum-starved MEFs were infected with an MOI = 10 of the indicated parasites for 18 h. Activation of Stat3 was also determined by Western blot analysis of cell extracts using anti–phospho-Stat3 Tyr 705. Stat3 and actin levels are shown as loading controls. Data are representative of three (A and E) or two (B–D) independent experiments.

Mentions: ROP16 is highly polymorphic between type I (or III) and II parasites (Saeij et al., 2006, 2007). Given that the relative expression levels among the three strains from the quantitative RT-PCR data deposited in ToxoDB are similar (unpublished data), the polymorphism might be responsible for the defective Stat3 activation in cells infected with type II parasites. However, the precise molecular mechanism involved remains to be elucidated. To address this issue, we cloned type II (ME49) ROP16 complementary DNA from a type II strain, ME49, into a mammalian expression vector. In 293T cells expressing ME49 ROP16, activation of the Stat3-dependent promoters was only slightly induced, as compared with cells expressing kinase-inactive ROP16D539A. However, it was dramatically lower compared with cells expressing type I (RH) ROP16, indicating that potency of ME49 ROP16 in Stat3 activation may be profoundly but not totally impaired (Fig. 4 A). Next, to identify which regions of the ME49 ROP16 protein are responsible for the defective Stat3 activation, a series of chimeric ROP16 constructs were generated. Domains of ROP16 were swapped between RH and ME49 ROP16, taking advantage of the presence of conserved restriction enzyme sites, and the resulting chimeras were assessed for Stat3 activation in 293T cells (Fig. 4 B). The results revealed a severe defect in Stat3 activation conferred by replacement of the central domain R2 in RH ROP16 with M2 of ME49 ROP16. This region exhibits nine polymorphic amino acid substitutions, and each of them were replaced in RH ROP16 with the corresponding sequence of ME49 and tested for Stat3-dependent reporter activation (Saeij et al., 2007; Fig. S4). Among them, the substitution of a leucine residue at position 503 to a serine (L503S) located in the kinase domain resulted in a dramatic decrease in Stat3 activation to a level comparable to that mediated by ME49 ROP16 (Fig. 4 C). Next, we assessed whether the single reverse mutation of the serine residue at position 503 to a leucine (S503L) in ME49 ROP16 restored Stat3 activation. We found that ME49 ROP16 S503L resulted in full restoration of Stat3 activation (Fig. 4 D). These findings indicated that a single amino acid of ROP16 may determine the strain difference between RH and ME49 strains. Finally, we generated transgenic parasite strains expressing WT or L503S-type RH ROP16 (RHWT or RHL503S, respectively) or WT or S503L-type ME49 ROP16 (ME49WT or ME49S503L, respectively) in rop16 KO parasites to assess their Stat3 activation in vivo (Fig. S3 B). In perfect agreement with a previous observation (Saeij et al., 2007), Stat3 activation, as measured by phosphorylation, was significantly increased by RHWT and ME49S503L and induced, but markedly reduced, by RHL503S or ME49WT (Fig. 4 E). These results indicate that WT ME49 (type II) ROP16 and L503S-type RH (type I) ROP16 mutants are impaired but not completely defective in Stat3 activation in vivo. Collectively, these results clearly demonstrated that the single amino acid polymorphism at position 503 in ROP16 determines the strain difference between type I and II strains in terms of Stat3 activation.


A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3.

Yamamoto M, Standley DM, Takashima S, Saiga H, Okuyama M, Kayama H, Kubo E, Ito H, Takaura M, Matsuda T, Soldati-Favre D, Takeda K - J. Exp. Med. (2009)

The L503S polymorphic mutation in type II ROP16 determines its defective Stat3 activation. (A–D) 293T cells were transfected with the APRE-luc plasmid together with the indicated ROP16 expression vectors. Luciferase activities were expressed as fold increases over the background levels shown by lysates prepared from mock-transfected cells. Indicated values are means ± the variation range of duplicates. A list of the chimeric ROP16 expression vectors is shown (B, left). R, RH (pink); M, ME49 (blue). (E) Serum-starved MEFs were infected with an MOI = 10 of the indicated parasites for 18 h. Activation of Stat3 was also determined by Western blot analysis of cell extracts using anti–phospho-Stat3 Tyr 705. Stat3 and actin levels are shown as loading controls. Data are representative of three (A and E) or two (B–D) independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806617&req=5

fig4: The L503S polymorphic mutation in type II ROP16 determines its defective Stat3 activation. (A–D) 293T cells were transfected with the APRE-luc plasmid together with the indicated ROP16 expression vectors. Luciferase activities were expressed as fold increases over the background levels shown by lysates prepared from mock-transfected cells. Indicated values are means ± the variation range of duplicates. A list of the chimeric ROP16 expression vectors is shown (B, left). R, RH (pink); M, ME49 (blue). (E) Serum-starved MEFs were infected with an MOI = 10 of the indicated parasites for 18 h. Activation of Stat3 was also determined by Western blot analysis of cell extracts using anti–phospho-Stat3 Tyr 705. Stat3 and actin levels are shown as loading controls. Data are representative of three (A and E) or two (B–D) independent experiments.
Mentions: ROP16 is highly polymorphic between type I (or III) and II parasites (Saeij et al., 2006, 2007). Given that the relative expression levels among the three strains from the quantitative RT-PCR data deposited in ToxoDB are similar (unpublished data), the polymorphism might be responsible for the defective Stat3 activation in cells infected with type II parasites. However, the precise molecular mechanism involved remains to be elucidated. To address this issue, we cloned type II (ME49) ROP16 complementary DNA from a type II strain, ME49, into a mammalian expression vector. In 293T cells expressing ME49 ROP16, activation of the Stat3-dependent promoters was only slightly induced, as compared with cells expressing kinase-inactive ROP16D539A. However, it was dramatically lower compared with cells expressing type I (RH) ROP16, indicating that potency of ME49 ROP16 in Stat3 activation may be profoundly but not totally impaired (Fig. 4 A). Next, to identify which regions of the ME49 ROP16 protein are responsible for the defective Stat3 activation, a series of chimeric ROP16 constructs were generated. Domains of ROP16 were swapped between RH and ME49 ROP16, taking advantage of the presence of conserved restriction enzyme sites, and the resulting chimeras were assessed for Stat3 activation in 293T cells (Fig. 4 B). The results revealed a severe defect in Stat3 activation conferred by replacement of the central domain R2 in RH ROP16 with M2 of ME49 ROP16. This region exhibits nine polymorphic amino acid substitutions, and each of them were replaced in RH ROP16 with the corresponding sequence of ME49 and tested for Stat3-dependent reporter activation (Saeij et al., 2007; Fig. S4). Among them, the substitution of a leucine residue at position 503 to a serine (L503S) located in the kinase domain resulted in a dramatic decrease in Stat3 activation to a level comparable to that mediated by ME49 ROP16 (Fig. 4 C). Next, we assessed whether the single reverse mutation of the serine residue at position 503 to a leucine (S503L) in ME49 ROP16 restored Stat3 activation. We found that ME49 ROP16 S503L resulted in full restoration of Stat3 activation (Fig. 4 D). These findings indicated that a single amino acid of ROP16 may determine the strain difference between RH and ME49 strains. Finally, we generated transgenic parasite strains expressing WT or L503S-type RH ROP16 (RHWT or RHL503S, respectively) or WT or S503L-type ME49 ROP16 (ME49WT or ME49S503L, respectively) in rop16 KO parasites to assess their Stat3 activation in vivo (Fig. S3 B). In perfect agreement with a previous observation (Saeij et al., 2007), Stat3 activation, as measured by phosphorylation, was significantly increased by RHWT and ME49S503L and induced, but markedly reduced, by RHL503S or ME49WT (Fig. 4 E). These results indicate that WT ME49 (type II) ROP16 and L503S-type RH (type I) ROP16 mutants are impaired but not completely defective in Stat3 activation in vivo. Collectively, these results clearly demonstrated that the single amino acid polymorphism at position 503 in ROP16 determines the strain difference between type I and II strains in terms of Stat3 activation.

Bottom Line: We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages.In addition, kinase-inactive ROP16 failed to activate Stat3.These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Graduate School of Medicine, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT
Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

Show MeSH
Related in: MedlinePlus