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

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).

In vitro activity, safety and in vivo efficacy of the novel bumped kinase inhibitor BKI-1748 in non-pregnant and pregnant mice experimentally infected with Neospora caninum tachyzoites and Toxoplasma gondii oocysts

“…Ultrathin sections were placed onto formvar-carbon-coated grids (PLANO, Wetzlar, Germany), and samples were observed on a Philips CM12 TEM operating at 80 kV. For SEM, MNCs and tachyzoites were separated from host cell debris by Sephadex-G-25-chromatography and were fixed and processed as described ( Winzer et al, 2020c ). They were placed onto glass coverslips, sputter-coated with gold and inspected on a Zeiss Gemini450 SEM operating at 5 kV.…”

The Impact of BKI-1294 Therapy in Mice Infected With the Apicomplexan Parasite Neospora caninum and Re-infected During Pregnancy

“…LC-MS and data base mining showed that these bands were comprised of several proteins with potentially upregulated expression in MNCs. However, recent comparative proteomics of N. caninum tachyzoites and MNCs did not pick up any of these proteins as differentially expressed in the two stages (22), indicating that their expression levels might be below the resolution limit of the shotgun proteomics approach. In any case, these proteins represent interesting candidates for the further characterization of MNCs and for studies on how this druginduced MNC stage could interact with the host.…”

“…A likely scenario is that MNCs were formed also in vivo, and this could be responsible for the observed reactivation of the live-vaccine and the increased vertical transmission upon re-infection. Shotgun proteomics had shown that MNCs exhibited an altered proteome when compared to tachyzoites, and this drug-induced stage was termed baryzoite (22). Following in vitro treatment with BKI-1294 during 5 days, it took approximately 10 days of culture without drug for MNCs to disappear and tachyzoites with SAG1 surface expression to re-emerge (21).…”

“…Tachyzoite and MNC preparations were done essentially as described (22). In short, semi-confluent BALB/c dermal fibroblast monolayers were maintained in T175 cell culture flasks infected with 10 6 Nc-Liv tachyzoites and passaged at least twice prior to the experiment.…”

“…BKI-1294 also affects the intracellular tachyzoites by inducing the formation of multinucleated complexes (MNCs) (20), which are characterized by continued nuclear division, and the formation of intracellular zoites separated by the inner membrane complex, but lacking the major surface antigen 1 (SAG1) (21). However, these zoites failed to undergo disjunction and tachyzoite formation, remained trapped within the host cell cytoplasm, and exhibited altered mRNA and protein expression (19,22). MNCs remained viable for up to 20 days under constant BKI-1294 drug pressure in vitro, and upon drug removal the block in zoite disjunction was alleviated and new infective tachyzoites were formed, which resumed proliferation (21).…”