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Ectopic expression of a Neospora caninum Kazal type inhibitor triggers developmental defects in Toxoplasma and Plasmodium.

Tampaki Z, Mwakubambanya RS, Goulielmaki E, Kaforou S, Kim K, Waters AP, Carruthers VB, Siden-Kiamos I, Loukeris TG, Koussis K - PLoS ONE (2015)

Bottom Line: NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress.Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes.Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece; Department of Biology, University of Crete, Heraklion, Greece.

ABSTRACT
Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characterization of NcPIS_6 strain.(A) Invasion competence of NcPIS_6 clone was similar to the RH strain. BAPTA-AM treated parasites, which do not invade cells, were included as a control. Error bars represent SEM (n = 3, each experiment was performed in duplicate and ten random fields were screened blindly in each sample). (B) Percentage of lysed vacuoles of NcPIS_6 versus RH after inducing egress with calcium ionophore A23187. Intracellular replicating tachyzoites (36–40 h p.i.) were treated with 1 μM A23187 or DMSO (solvent control) for 5min. Percentage of induced egress from host cells was determined. Error bars, SEM (n = 3, each experiment was performed in duplicate and, 10 fields were blindly screened per sample). (C) Replication assay of NcPIS_6, RH and RHΔhxgprt- (parental) and (D) NcPISmut_16 and RHΔhxgprt-. Following a 24 h interval of intracellular replication, vacuoles from all the above strains were scored according to their parasite context. Error bars, SEM (n = 3, representative of duplicate samples); P<0.001, Student’s t-test.
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pone.0121379.g003: Phenotypic characterization of NcPIS_6 strain.(A) Invasion competence of NcPIS_6 clone was similar to the RH strain. BAPTA-AM treated parasites, which do not invade cells, were included as a control. Error bars represent SEM (n = 3, each experiment was performed in duplicate and ten random fields were screened blindly in each sample). (B) Percentage of lysed vacuoles of NcPIS_6 versus RH after inducing egress with calcium ionophore A23187. Intracellular replicating tachyzoites (36–40 h p.i.) were treated with 1 μM A23187 or DMSO (solvent control) for 5min. Percentage of induced egress from host cells was determined. Error bars, SEM (n = 3, each experiment was performed in duplicate and, 10 fields were blindly screened per sample). (C) Replication assay of NcPIS_6, RH and RHΔhxgprt- (parental) and (D) NcPISmut_16 and RHΔhxgprt-. Following a 24 h interval of intracellular replication, vacuoles from all the above strains were scored according to their parasite context. Error bars, SEM (n = 3, representative of duplicate samples); P<0.001, Student’s t-test.

Mentions: To study the effect of expressing NcPI-S and its mutated form in T. gondii tachyzoites we performed phenotypic characterization. NcPIS_6 tachyzoites were compared to WT tachyzoites of the RH and the parental RHΔhxgprt strains using well-established invasion [47], egress [48] and growth assays. Neither invasion (Fig. 3A) nor egress assays (Fig. 3B) revealed any significant phenotypic deviation of NcPIS_6 from the WT control RH strain (Fig. 3A and 3B). To assess growth we carried out replication assays [57] where equal numbers of tachyzoites from NcPIS_6 and WT control strains (RH and RHΔhxgprt), were used to infect HFF cells (Fig. 3C). At 24 h p.i., vacuoles derived from control strains contained 8 tachyzoites in a percentage ranging between 40–70% of the total vacuoles, indicating 3 cell divisions as was expected. In contrast, only ∼17% of NcPIS_6 vacuoles contained 8 tachyzoites (Fig. 3C), indicating impaired replication. A clone (NcPIS_8), derived from a second independent transformation experiment, showed similar growth kinetics to that of the NcPIS_6 (S1A Fig.), verifying that a replication defect of T. gondii tachyzoites is reproducibly associated with the expression of (myc)NcPI-S. In contrast NcPISmut_16 tachyzoites showed the same growth dynamics as the parental RHΔhxgprt strain (Fig. 3D). When combined, these results suggest that active NcPI-S is expressed in tachyzoites, and interfere with proliferation of the parasite, while the mutated form has no effect on parasite growth.


Ectopic expression of a Neospora caninum Kazal type inhibitor triggers developmental defects in Toxoplasma and Plasmodium.

Tampaki Z, Mwakubambanya RS, Goulielmaki E, Kaforou S, Kim K, Waters AP, Carruthers VB, Siden-Kiamos I, Loukeris TG, Koussis K - PLoS ONE (2015)

Phenotypic characterization of NcPIS_6 strain.(A) Invasion competence of NcPIS_6 clone was similar to the RH strain. BAPTA-AM treated parasites, which do not invade cells, were included as a control. Error bars represent SEM (n = 3, each experiment was performed in duplicate and ten random fields were screened blindly in each sample). (B) Percentage of lysed vacuoles of NcPIS_6 versus RH after inducing egress with calcium ionophore A23187. Intracellular replicating tachyzoites (36–40 h p.i.) were treated with 1 μM A23187 or DMSO (solvent control) for 5min. Percentage of induced egress from host cells was determined. Error bars, SEM (n = 3, each experiment was performed in duplicate and, 10 fields were blindly screened per sample). (C) Replication assay of NcPIS_6, RH and RHΔhxgprt- (parental) and (D) NcPISmut_16 and RHΔhxgprt-. Following a 24 h interval of intracellular replication, vacuoles from all the above strains were scored according to their parasite context. Error bars, SEM (n = 3, representative of duplicate samples); P<0.001, Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372514&req=5

pone.0121379.g003: Phenotypic characterization of NcPIS_6 strain.(A) Invasion competence of NcPIS_6 clone was similar to the RH strain. BAPTA-AM treated parasites, which do not invade cells, were included as a control. Error bars represent SEM (n = 3, each experiment was performed in duplicate and ten random fields were screened blindly in each sample). (B) Percentage of lysed vacuoles of NcPIS_6 versus RH after inducing egress with calcium ionophore A23187. Intracellular replicating tachyzoites (36–40 h p.i.) were treated with 1 μM A23187 or DMSO (solvent control) for 5min. Percentage of induced egress from host cells was determined. Error bars, SEM (n = 3, each experiment was performed in duplicate and, 10 fields were blindly screened per sample). (C) Replication assay of NcPIS_6, RH and RHΔhxgprt- (parental) and (D) NcPISmut_16 and RHΔhxgprt-. Following a 24 h interval of intracellular replication, vacuoles from all the above strains were scored according to their parasite context. Error bars, SEM (n = 3, representative of duplicate samples); P<0.001, Student’s t-test.
Mentions: To study the effect of expressing NcPI-S and its mutated form in T. gondii tachyzoites we performed phenotypic characterization. NcPIS_6 tachyzoites were compared to WT tachyzoites of the RH and the parental RHΔhxgprt strains using well-established invasion [47], egress [48] and growth assays. Neither invasion (Fig. 3A) nor egress assays (Fig. 3B) revealed any significant phenotypic deviation of NcPIS_6 from the WT control RH strain (Fig. 3A and 3B). To assess growth we carried out replication assays [57] where equal numbers of tachyzoites from NcPIS_6 and WT control strains (RH and RHΔhxgprt), were used to infect HFF cells (Fig. 3C). At 24 h p.i., vacuoles derived from control strains contained 8 tachyzoites in a percentage ranging between 40–70% of the total vacuoles, indicating 3 cell divisions as was expected. In contrast, only ∼17% of NcPIS_6 vacuoles contained 8 tachyzoites (Fig. 3C), indicating impaired replication. A clone (NcPIS_8), derived from a second independent transformation experiment, showed similar growth kinetics to that of the NcPIS_6 (S1A Fig.), verifying that a replication defect of T. gondii tachyzoites is reproducibly associated with the expression of (myc)NcPI-S. In contrast NcPISmut_16 tachyzoites showed the same growth dynamics as the parental RHΔhxgprt strain (Fig. 3D). When combined, these results suggest that active NcPI-S is expressed in tachyzoites, and interfere with proliferation of the parasite, while the mutated form has no effect on parasite growth.

Bottom Line: NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress.Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes.Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece; Department of Biology, University of Crete, Heraklion, Greece.

ABSTRACT
Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage.

No MeSH data available.


Related in: MedlinePlus