title: Ectopic expression of a Neospora caninum Kazal type inhibitor triggers developmental defects in Toxoplasma and Plasmodium.
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.
authors: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece; Department of Biology, University of Crete, Heraklion, Greece.
authors: Zoi
authors: Z
authors: Tampaki
authors: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece; Department of Biology, University of Crete, Heraklion, Greece.
authors: Ramadhan S
authors: RS
authors: Mwakubambanya
authors: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece; Department of Biology, University of Crete, Heraklion, Greece.
authors: Evi
authors: E
authors: Goulielmaki
authors: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece.
authors: Sofia
authors: S
authors: Kaforou
authors: Department of Medicine, Albert Einstein College of Medicine, Bronx, United States of America.
authors: Kami
authors: K
authors: Kim
authors: Faculty of Biomedical & Life Sciences, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, Scotland, United Kingdom.
authors: Andrew P
authors: AP
authors: Waters
authors: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, United States of America.
authors: Vern B
authors: VB
authors: Carruthers
authors: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece.
authors: Inga
authors: I
authors: Siden-Kiamos
authors: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece.
authors: Thanasis G
authors: TG
authors: Loukeris
authors: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece.
authors: Konstantinos
authors: K
authors: Koussis
mesh_terms: D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods; D055808:Drug Discovery / Q000379:methods; D000066630:Ectopic Gene Expression / Q000502:physiology; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D046529:Microscopy, Electron, Transmission; D018688:Neospora / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D010962:Plasmodium berghei / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D059748:Proteolysis; D015842:Serine Proteinase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D014122:Toxoplasma / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism; D014162:Transfection
chemical_list: D017931:DNA Primers; D015842:Serine Proteinase Inhibitors