Neospora caninum: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294.

Pablo Winzer,Wesley C Van Voorhis,Manfred Heller,Dennis Imhof,Luis-Miguel Ortega-Mora,Andrew Hemphill,Joachim Müller,Dominic Ritler,Kayode K Ojo,Anne-Christine Uldry,Sophie Braga-Lagache

doi:10.3390/microorganisms8060801

Pablo Winzer, Wesley C Van Voorhis + Show 9 more

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https://doi.org/10.3390/microorganisms8060801

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Abstract

Background: the apicomplexan parasite Neospora caninum causes important reproductive problems in farm animals, most notably in cattle. After infection via oocysts or tissue cysts, rapidly dividing tachyzoites infect various tissues and organs, and in immunocompetent hosts, they differentiate into slowly dividing bradyzoites, which form tissue cysts and constitute a resting stage persisting within infected tissues. Bumped kinase inhibitors (BKIs) of calcium dependent protein kinase 1 are promising drug candidates for the treatment of Neospora infections. BKI-1294 exposure of cell cultures infected with N. caninum tachyzoites results in the formation of massive multinucleated complexes (MNCs) containing numerous newly formed zoites, which remain viable for extended periods of time under drug pressure in vitro. MNC and tachyzoites exhibit considerable antigenic and structural differences. Methods: Using shotgun mass spectrometry, we compared the proteomes of tachyzoites to BKI-1294 induced MNCs, and analyzed the mRNA expression levels of selected genes in both stages. Results: More than half of the identified proteins are downregulated in MNCs as compared to tachyzoites. Only 12 proteins are upregulated, the majority of them containing SAG1 related sequence (SRS) domains, and some also known to be expressed in bradyzoites Conclusions: MNCs exhibit a proteome different from tachyzoites, share some bradyzoite-like features, but may constitute a third stage, which remains viable and ensures survival under adverse conditions such as drug pressure. We propose the term “baryzoites” for this stage (from Greek βαρυσ = massive, bulky, heavy, inert).

Highlights

The phylum Apicomplexa parasites includes important pathogens for humans and animals
The proteome dataset generated for this study, comparing protein expression in Bumped kinase inhibitors (BKIs)-1294-induced multinucleated complexes (MNCs) and tachyzoites, contained 2558 non-redundant proteins, corresponding to 36% of the 7121 open reading frames identified in the N. caninum genome [24], nearly two times more than the number of proteins identified in a previous study [20]
This suggests that MNCs may represent a resting state of the parasite that is induced by drug pressure exerted through BKI-1294 treatment

Summary

Introduction

The phylum Apicomplexa parasites includes important pathogens for humans and animals. Canines act as definitive hosts of N. caninum, and sexual development of the parasite in the intestinal tissue leads to the formation of oocysts, which are shed with the feces. Canines can act as intermediate hosts and are affected by neurological symptoms [2]. Three stages are important for the life cycle of N. caninum, sporozoites, tachyzoites, and bradyzoites. Tachyzoites represent the rapidly proliferating disease-causing stage, which infects numerous cell types and tissues, and can cross the placenta and affect the developing fetus, which leads to malformations and/or abortion. The slowly proliferating bradyzoites are formed upon the immunological and physiological host response, and they represent a tissue cyst-forming stage that persists within infected tissues for extended periods. A temporal immunomodulation, such as during pregnancy, frequently leads to recrudescence and re-differentiation into tachyzoites which infect the developing fetus [3]. There is a keen interest in novel chemotherapeutics acting on specific targets [6]

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