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Neospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294

1
Institute of Parasitology, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland
2
Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland
3
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria, 28040 Madrid, Spain
4
Center for Emerging and Re-emerging Infectious Diseases (CERID), Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA 98109, USA
*
Author to whom correspondence should be addressed.
Pathogens 2020, 9(5), 382; https://doi.org/10.3390/pathogens9050382
Received: 20 April 2020 / Revised: 11 May 2020 / Accepted: 12 May 2020 / Published: 16 May 2020
(This article belongs to the Special Issue Neospora Caninum: Infection and Immunity)
Background: Bumped kinase inhibitors (BKIs) are potential drugs for neosporosis treatment in farm animals. BKI-1294 exposure results in the formation of multinucleated complexes (MNCs), which remain viable in vitro under constant drug pressure. We investigated the formation of BKI-1294 induced MNCs, the re-emergence of viable tachyzoites following drug removal, and the localization of CDPK1, the molecular target of BKIs. Methods: N. caninum tachyzoites and MNCs were studied by TEM and immunofluorescence using antibodies directed against CDPK1, and against NcSAG1 and IMC1 as markers for tachyzoites and newly formed zoites, respectively. Results: After six days of drug exposure, MNCs lacked SAG1 surface expression but remained intracellular, and formed numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective tachyzoites after 10 days. In intracellular tachyzoites, CDPK1 was evenly distributed but shifted towards the apical part once parasites were extracellular. This shift was not affected by BKI-1294. Conclusions: CDPK1 has a dynamic distribution depending on whether parasites are located within a host cell or outside. During MNC-to-tachyzoite reconversion newly formed tachyzoites are generated directly from MNCs through zoites of unknown surface antigen composition. Further in vivo studies are needed to determine if MNCs could lead to a persistent reservoir of infection after BKI treatment. View Full-Text
Keywords: bumped kinase inhibitor; calcium-dependent protein kinase; immunofluorescence; immunogold labeling; inner membrane complex; neosporosis; surface antigen; tachyzoite; transmission electron microscopy bumped kinase inhibitor; calcium-dependent protein kinase; immunofluorescence; immunogold labeling; inner membrane complex; neosporosis; surface antigen; tachyzoite; transmission electron microscopy
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Winzer, P.; Anghel, N.; Imhof, D.; Balmer, V.; Ortega-Mora, L.-M.; Ojo, K.K.; Van Voorhis, W.C.; Müller, J.; Hemphill, A. Neospora caninum: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294. Pathogens 2020, 9, 382.

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