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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

Mature NcPI-S_3 ookinetes contain fewer micronemes.(A) Selected longitudinal sections, across the apical part of wild type (left image) and NcPIS_3 mature ookinetes (middle and right images). All are surrounded by the characteristic subpellicular inner membrane complex. Scarcity of micronemes (white arrows), is a common feature in NcPIS_3 ookinetes. The ookinete-specific characteristic structure of crystalloid (cr) is clearly visible. (B) (Left panel) Semi-quantitative RT-PCR analysis of micronemal proteins CTRP and SOAP in NcPIS_3 and GFPp ookinetes. No alteration was observed in transcript levels. p28 was used as a control. (Right panel) Western analysis of soluble (SF) and membrane (MF) fraction of ookinete extracts derived from in vitro cultured NcPIS_3, NcPISmut and GFPp ookinetes, with antibodies against the micronemal protein SOAP. P28 was used as a sample normalization control. No SOAP is detected in the NcPIS_3 line. (C) Western blot analysis of PbeIF2a phosphorylation. Western blots loaded with lysates from GFPp, NcPIS_3 and NcPISmut ookinete cultures were probed with antibodies against phosphorylated eIF2α. NcPIS_3 parasites show an increase in PbeIF2a phosphorylation. The endoplasmic reticulum (ER) marker, BiP was used as the loading control.
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pone.0121379.g006: Mature NcPI-S_3 ookinetes contain fewer micronemes.(A) Selected longitudinal sections, across the apical part of wild type (left image) and NcPIS_3 mature ookinetes (middle and right images). All are surrounded by the characteristic subpellicular inner membrane complex. Scarcity of micronemes (white arrows), is a common feature in NcPIS_3 ookinetes. The ookinete-specific characteristic structure of crystalloid (cr) is clearly visible. (B) (Left panel) Semi-quantitative RT-PCR analysis of micronemal proteins CTRP and SOAP in NcPIS_3 and GFPp ookinetes. No alteration was observed in transcript levels. p28 was used as a control. (Right panel) Western analysis of soluble (SF) and membrane (MF) fraction of ookinete extracts derived from in vitro cultured NcPIS_3, NcPISmut and GFPp ookinetes, with antibodies against the micronemal protein SOAP. P28 was used as a sample normalization control. No SOAP is detected in the NcPIS_3 line. (C) Western blot analysis of PbeIF2a phosphorylation. Western blots loaded with lysates from GFPp, NcPIS_3 and NcPISmut ookinete cultures were probed with antibodies against phosphorylated eIF2α. NcPIS_3 parasites show an increase in PbeIF2a phosphorylation. The endoplasmic reticulum (ER) marker, BiP was used as the loading control.

Mentions: Careful inspection of the TEM pictures revealed that the few mature NcPIS_3 ookinetes which developed, suffered from ultra-structural malformations including a severe decrease in the number of micronemes (Fig. 6A). To further investigate this we chose to study the micronemal protein SOAP. We first verified that the soap transcript levels in the NcPIS_3 zygotes/ookinetes were comparable to WT (Fig. 6B left). However, Western blot analysis showed that the micronemal protein SOAP [60] was hardly detectable in NcPIS_3 zygote/ookinete extracts, compared to GFPp or NcPISmut derived extracts where this protein was readily seen (Fig. 6B right). Furthermore we repeated these experiments using the NcPIS_1 parasite clone to exclude any effects deriving from the genetic background. The results were identical (S5 Fig.).


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)

Mature NcPI-S_3 ookinetes contain fewer micronemes.(A) Selected longitudinal sections, across the apical part of wild type (left image) and NcPIS_3 mature ookinetes (middle and right images). All are surrounded by the characteristic subpellicular inner membrane complex. Scarcity of micronemes (white arrows), is a common feature in NcPIS_3 ookinetes. The ookinete-specific characteristic structure of crystalloid (cr) is clearly visible. (B) (Left panel) Semi-quantitative RT-PCR analysis of micronemal proteins CTRP and SOAP in NcPIS_3 and GFPp ookinetes. No alteration was observed in transcript levels. p28 was used as a control. (Right panel) Western analysis of soluble (SF) and membrane (MF) fraction of ookinete extracts derived from in vitro cultured NcPIS_3, NcPISmut and GFPp ookinetes, with antibodies against the micronemal protein SOAP. P28 was used as a sample normalization control. No SOAP is detected in the NcPIS_3 line. (C) Western blot analysis of PbeIF2a phosphorylation. Western blots loaded with lysates from GFPp, NcPIS_3 and NcPISmut ookinete cultures were probed with antibodies against phosphorylated eIF2α. NcPIS_3 parasites show an increase in PbeIF2a phosphorylation. The endoplasmic reticulum (ER) marker, BiP was used as the loading control.
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Related In: Results  -  Collection

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

pone.0121379.g006: Mature NcPI-S_3 ookinetes contain fewer micronemes.(A) Selected longitudinal sections, across the apical part of wild type (left image) and NcPIS_3 mature ookinetes (middle and right images). All are surrounded by the characteristic subpellicular inner membrane complex. Scarcity of micronemes (white arrows), is a common feature in NcPIS_3 ookinetes. The ookinete-specific characteristic structure of crystalloid (cr) is clearly visible. (B) (Left panel) Semi-quantitative RT-PCR analysis of micronemal proteins CTRP and SOAP in NcPIS_3 and GFPp ookinetes. No alteration was observed in transcript levels. p28 was used as a control. (Right panel) Western analysis of soluble (SF) and membrane (MF) fraction of ookinete extracts derived from in vitro cultured NcPIS_3, NcPISmut and GFPp ookinetes, with antibodies against the micronemal protein SOAP. P28 was used as a sample normalization control. No SOAP is detected in the NcPIS_3 line. (C) Western blot analysis of PbeIF2a phosphorylation. Western blots loaded with lysates from GFPp, NcPIS_3 and NcPISmut ookinete cultures were probed with antibodies against phosphorylated eIF2α. NcPIS_3 parasites show an increase in PbeIF2a phosphorylation. The endoplasmic reticulum (ER) marker, BiP was used as the loading control.
Mentions: Careful inspection of the TEM pictures revealed that the few mature NcPIS_3 ookinetes which developed, suffered from ultra-structural malformations including a severe decrease in the number of micronemes (Fig. 6A). To further investigate this we chose to study the micronemal protein SOAP. We first verified that the soap transcript levels in the NcPIS_3 zygotes/ookinetes were comparable to WT (Fig. 6B left). However, Western blot analysis showed that the micronemal protein SOAP [60] was hardly detectable in NcPIS_3 zygote/ookinete extracts, compared to GFPp or NcPISmut derived extracts where this protein was readily seen (Fig. 6B right). Furthermore we repeated these experiments using the NcPIS_1 parasite clone to exclude any effects deriving from the genetic background. The results were identical (S5 Fig.).

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