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

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).

(high virulence) during different steps of the lytic cycle [20]. To investigate BKI-1294-induced MNCs in more detail, we thus compared the proteomes of isolated tachyzoites to the proteomes of MNCs by shotgun mass spectrometry.

Tissue Culture Media, Biochemicals, and Drugs
If not stated otherwise, all tissue culture media were purchased from Gibco-BRL (Zürich, Switzerland), and biochemicals from Sigma (St. Louis, MO, USA). BKI-1294 was synthesized to >99% purity by HPLC and NMR by Wuxi AppTech, Inc. (Wuhan, China) and provided by the Center for Emerging and Reemerging Infectious Diseases (CERID), Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington (Seattle, WA, USA), and was stored in powder form, dry and protected from light at room temperature. For in vitro studies, 20 mM stock solutions were prepared in dimethyl sulfoxide (DMSO) and they were stored at −20 • C. Primers for real-time PCR (RT-PCR) were purchased from Eurofins (Luxemburg, Luxemburg).

Microscopy
Transmission electron microscopy (TEM) of BKI-1294-treated or non-treated Nc-Spain7-infected HFF was done as previously described [12,17]. In short, infected monolayers grown in T-25 tissue culture flasks were fixed in 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer pH 7.3, removed from the flask by scraping, post-fixed in 2% osmium tetroxide in cacodylate buffer, and sequentially dehydrated in a graded (30-50-70-90-100%) ethanol series. Specimens were embedded in Epon 812 epoxy resin, polymerized at 65 • C, and ultrathin sections were cut on a Reichert and Jung ultramicrotome (Reichert & Jung, Vienna). Following transfer onto 300 mesh formvar-carbon coated nickel grids (Plano GmbH, Marburg, Germany), they were stained in Uranyless™ and lead citrate (EMS, Hatfield, PA, USA), and specimens were inspected on a CM12 TEM operating at 80 kV.
For scanning electron microscopy (SEM), semi-confluent HFF monolayers were maintained in T175 cell culture flasks and were infected with Nc-Spain7 tachyzoites. At 4 h post infection, treatments with 5 µM BKI-1294, or the corresponding amount of DMSO in controls were initiated, and cultures were maintained at 37 • C/5% CO 2 during 3 (controls) or 6 days (BKI-1294-treated cultures), until MNCs were formed. Subsequently, the infected monolayers were washed twice with PBS, followed by removal of infected cells with a rubber cell scraper and resuspension in PBS. After passaging the suspensions twice through a 25-gauge needle to break the host cells, MNCs and tachyzoites were separated from host cell debris by Sephadex G-25 chromatography. The parasite fractions in the flow through were collected by centrifugation (15 min, 1000× g, 4 • C), washed twice with PBS, and were fixed in 2.5% glutaraldehyde and 2% osmium tetroxide, and dehydrated in ethanol as for TEM. They were finally washed twice in hexamethyl-disilazane (HDS), taken up in a small volume of HDS, and were allowed to settle onto glass coverslips under a fume hood. They were then sputter-coated with gold and inspected on a JEOL 840 SEM operating at 25 kV.

Proteomics
Semi-confluent HFF monolayers were maintained in T175 cell culture flasks, and were infected with Nc-Spain7 tachyzoites. At 4 h post infection, treatment with 5 µM BKI-1294, or the corresponding amount of DMSO in controls, was initiated, and cultures were maintained at 37 • C/5% CO 2 during 3 (controls) or 6 days (BKI-1294-treated cultures), until multinucleated complexes (MNCs) were formed. Subsequently, the infected monolayers were washed twice with PBS, followed by removal of infected cells with a rubber cell scraper and resuspension in PBS. After passaging the suspensions twice through a 25 gauge needle to break the host cells, MNCs and tachyzoites were separated from host cell debris by Sephadex G-25 chromatography. The parasite fractions in the flow through were collected by centrifugation (15 min, 1000× g, 4 • C) and washed twice with PBS, as described [21].
Cell pellets were lysed in 100 µL 8M urea/100 mM Tris/HCl pH 8/cOmplete TM protease inhibitor cocktail (Roche Diagnostics, Rotkreuz, Switzerland) by incubation for 15 min at RT followed by 15 min in an ultrasonic water bath. Proteins were reduced and alkylated with 10 mM DTT for 30 min at 37 • C and 50 mM iodoacetamide for 30 min at 37 • C. Proteins were precipitated at −20 • C by addition of 5 volumes cold acetone and incubation at −20 • C overnight. All liquid was carefully removed, and the pellet dried in ambient air for 15 min before reconstitution of proteins in 200 µL of 8 M urea, 50 mM Tris-HCl pH 8.0. Protein concentration was determined by Bradford assay and an aliquot corresponding to 10 µg protein was digested by trypsin (1:50 trypsin/protein ratio) for 6 hours at 37 • C after dilution of urea concentration to 1.6 M with 20 mM Tris-HCl pH 8.0 and 2 mM CaCl 2 . The digests were acidified with TFA (1%) and analyzed by LC-MS/MS. Three repetitive injections of an aliquot corresponding to 500 ng of protein digest was separated on an EASY-nLC 1000 coupled to a QExactive mass spectrometer (ThermoFisher Scientific). Peptides were trapped on an Acclaim PepMap100 C18 pre-column (3 µm, 100 Å, 75 µm × 2 cm, ThermoFisher Scientific, Reinach, Switzerland) and separated by backflush on a C18 column (3 µm, 100 Å, 75 µm × 15 cm, Nikkyo Technos, Tokyo, Japan) by applying a 60 min gradient of 5% to 40% acetonitrile in water and 0.1% formic acid, at a flow rate of 400 nL/min. Peptides of m/z 360-1400 were detected with resolution of 70,000, applying an automatic gain control (AGC) target of 10 6 and a maximum ion injection time of 50 ms. A top ten data dependent method for precursor ion fragmentation with a stepped 27% normalized collision energy was applied with the following settings: precursor isolation width of 2 m/z, resolution 17,500, AGC of 10 5 with a minimum target of 10 3 , maximum ion time of 110 ms, charge exclusion of unassigned and 1+ ions, peptide match on, and dynamic exclusion for 20 s, respectively.

Measurement of RNA Expression Levels by Quantitative Reverse Transcriptase Real-Time PCR
To determine the RNA levels of selected N. caninum genes (see Table 1), quantitative reverse transcriptase RT-PCR was performed as described [12,22]. Briefly, 8 × 10 4 HFF/well were seeded onto 6 well plates and cultured for 4 days at 37 • C/5% CO 2 prior to infection with 6 × 10 5 N. caninum tachyzoites/well. At 4 h post infection, the supernatant was aspirated and replaced with fresh medium containing either 5 µM BKI-1294 to form MNCs, or the corresponding amount of DMSO to generate tachyzoites. At 3 days post infection in the case of tachyzoites or 6 days post infection in the case of MNCs, RNA was isolated using the Nucleospin ® RNA Plus RNA isolation kit (Machery-Nagel, Düren, Germany) followed by cDNA synthesis using the Promega GoScript-Kit (Promega, Madison, WI, USA) according to the instructions provided by the manufacturers. Quantitative RT-PCR was performed using the Corbett Rotor gene 6000 (Qiagen, Venlo, Netherlands). The reactions contained 10 µL Sybr-Green-Mix, 0.1 µL of the forward and reverse primers listed in Table 1 and 10 µL of cDNA.

Statistics
The MS data were obtained from three biological replicates, with two technical replicates for each biological replicate. All MS data were processed by MaxQuant (version 1.5.4.1) with matching between runs for the same strain activated, but not between different strains, in order to avoid over-interpretation of the data. The sample sets were interpreted separately by MaxQuant. Fragment spectra were interpreted against a recent N. caninum protein sequence database in fasta format (ToxoDB-44_NcaninumLIV_AnnotatedProteins.fasta). The trypsin cleavage rule allowed amide bond cleavage after lysine and arginine but not if a proline follows, up to three missed cleavage sites, fixed carbamidomethylation modification of cysteines, variable oxidation of methionine and acetylation of protein N-termini. Precursor and fragment mass tolerances were set to 10 and 20 ppm, respectively. Peptide spectrum matches and peptide and protein group identifications were filtered to a 1% false discovery rate (FDR) based on reversed database sequence matches, and a minimum of two razor or unique peptides were required to accept a protein group identification. Protein identifications considered as contaminations (e.g., trypsin or BSA) as well as proteins identified only by site (considered by MaxQuant developers as very likely false positives) were removed for statistical validation. The normalized label-free quantification (LFQ) protein group intensities as calculated by MaxQuant and the sum of the three most intense peptide intensities were used for relative proteome quantifications. First, we imputed missing protein LFQ values for samples in any condition group when there were at least two LFQ intensities in one group (downshift of 1.8 S.D. with a width of 0.3 S.D. [23]). Before summing, missing peptide intensities were imputed sample group wise, when there were at least 2 valid intensities (downshift of 1.8 S.D. with a width of 0.3 S.D.). The resulting protein intensities were named iTop3. This process left proteins without values in one or the other group. For Welch's T-tests, those missing protein intensities were replaced by imputed values from the very low end of intensity distributions in analogy to the above described imputation strategy (downshift 1.8 S.D., width of 0.3 S.D.). An FDR-controlled Benjamini-Hochberg procedure was used for correction of p-values. A log2-fold change of at least 1 and a corrected p-value of ≤0.05 were required to be considered as significant. Statistical testing and imputation were made with a homemade R (version 3.6.2 script run under R-Studio).

Formation of MNCs upon BKI-1294 Treatment In Vitro
Treatment of N. caninum infected HFF with 5 µM BKI-1294 lead to the formation of intracellular MNCs within 3-6 days, which are comparatively visualized with non-drug-treated tachyzoites in Figure 1. TEM of non-treated tachyzoites showed intracellular parasites undergoing endodyogeny, situated within a parasitophorous vacuole ( Figure 1A). Figure 1C shows an MNC that formed after 6 days of treatment with BKI-1294. These MNCs were also located within a parasitophorous vacuole, were structurally intact, viable, and exhibited the typical features of apicomplexan parasites, but were not able to disconnect from each other and thus remained intracellular. SEM was performed on MNCs and non-treated tachyzoites that were separated from host cell debris by Sephadex G25 chromatography ( Figure 1B,D). MNCs exhibited multiple newly formed apical complexes that emerged out of the MNC surface, as indicated in Figure 1D.

The Proteome of Multinucleated Complexes Is Different from the Proteome of Tachyzoites
Shotgun mass spectrometry of the proteomes of N. caninum tachyzoites and of BKI-induced MNCs resulted in the identification of 207,041 unique peptides matching to 2558 proteins ( Table 2). The complete dataset is given in Table S1, which is available online. Table 2. Summary of protein quantification data. N. caninum tachyzoites and MNCs were grown, purified and subjected to MS shotgun analysis as described in the Materials and Methods. For each group, three biological replicates have been tested (with 2 technical replicates per biological replicate). Only proteins with significant differences in both iTop3 and iLFQ levels were regarded as differential.

Parameter Number
Unique peptides 207041 Non-redundant proteins 2558 Higher in tachyzoites 1325 Higher in MNCs 12 Similar levels in both 1221 By deeper analysis, we identified 1337 proteins that were differential between both stages with respect to both iTop3 and iLFQ parameters, as explained in Section 2.6. The large majority had lower iLFQ and iTop3 levels in MNCs as compared to tachyzoites. Only 12 proteins had higher levels ( Table 2).
Unbiased analysis of the dataset by principal component analysis (PCA) demonstrated for both iTop3 and iLFQ values (see Section 2.6) that the proteomes of tachyzoites and BKI-1294 induced MNCs formed non-overlapping clusters separated by principal component 1 (PC1). Moreover, the proteomes of biological and technical replicates clustered together for MNCs whereas the proteomes of biological and technical replicates of tachyzoites were separated along PC2 (Figure 2).

Multinucleated Complexes Express a Distinct Set of Potential Surface Antigens
A closer look at the 12 proteins with higher levels in MNCs compared to tachyzoites revealed that seven proteins had N-terminally located signal peptides, and six proteins had one or more SRS-domains (Table 3).  The relative abundance of these 12 proteins given by their respective iLFQ intensities-negligible in tachyzoites-reached up to 10 8 iLFQ units in MNCs in the case of the SRS-domain containing protein NCLIV_019580, formerly annotated as SAG4 (Figure 3).  Table 3. For all proteins, mean values ± one standard deviation for LFQ intensities (×10 6 ) in three biological replicates are shown. The proteins are termed by their respective accession numbers in the ToxoDB.

The SRS Domain Containing Protein NCLIV_019580 (NcSAG4) Has Higher mRNA Expression Levels in MNCs Compared to Tachyzoites
Since the SRS-domain containing protein encoded by NCLIV_019580, also annotated as NcSAG4, had the highest differential protein level in MNCs (see Figure 2), we investigated whether the NcSAG4 protein levels were correlated to the respective mRNA expression levels. We compared the mRNA levels in tachyzoites and MNCs of the corresponding coding sequence to N. caninum 28S RNA and included three other transcripts, namely mRNA coding for BAG1, IMC1, and CDPK1, the target of BKI-1294, into this comparison. As shown in Figure 4, MNCs had higher NcSAG4 mRNA levels compared to tachyzoites. Cells were harvested, mRNA was extracted, cDNA was prepared, and mRNA levels were quantified as described in Section 2.5. Mean ± standard error values from four replicates were assessed and expression levels were given as values in arbitrary units relative to the amount of 28S rRNA. The genes are termed by their respective accession numbers in ToxoDB.
The same was found for the expression level of mRNA coding for NcBAG1. The absolute mRNA levels of BAG1 were, however, three magnitudes lower than those of the three other genes investigated. Conversely, the levels of mRNAs encoding CDPK1 and IMC1 were lower in MNCs than in tachyzoites.

Discussion
The proteome dataset generated for this study, comparing protein expression in BKI-1294-induced MNC and tachyzoites, contained 2558 non-redundant proteins, corresponding to 36% of the 7121 open reading frames identified in the N. caninum genome [24], thus nearly two times more than the number of proteins identified in a previous study [20].
In our comparative analysis of MNCs and tachyzoites, more than half of these proteins, including key enzymes of energy and intermediary metabolism (see Table S1 for details), are downregulated in MNCs induced by BKI-1294 treatment, and only twelve proteins are upregulated in MNCs. This suggests that MNCs may represent a resting state of the parasite that is induced by drug pressure exerted through BKI-1294 treatment.
This hypothesis is backed by the finding that mRNA coding for NcBAG1 (NCLIV_027470), also known as a heat-shock protein and a well-established marker for bradyzoites [25], can be detected in MNCs, but not in tachyzoites. The same has not only been observed in MNCs induced by BKI-1294 in a previous study [12], but also in intracellular parasites exposed to a different class of compounds, namely artemisinin derivatives such as artemisone and GC012 [22]. In addition, these findings were confirmed on the protein level by observing increased anti-BAG1 antibody staining in HFF infected with N. caninum and treated with these compounds [22]. The corresponding NcBAG1 protein is, however, not included in the proteome dataset (see Table S1), suggesting that its expression levels are below the resolution limit of the method. Conversely, two other bradyzoite antigens, namely the SRS domain containing proteins NCLIV_010030, the bradyzoite antigen NcBSR4 [26], and NCLIV_019580, formerly annotated as NcSAG4, are amongst the twelve MNC-upregulated proteins. Of these, NcSAG4 exhibits the highest expression levels.
The N. caninum SRS domain containing protein superfamily comprises 227 genes and 52 pseudogenes [24], considerably more than Toxoplasma gondii strains [27]. The most prominent of these proteins is certainly SAG1 (NCLIV_033230), the major surface antigen of N. caninum. During the last two decades, NcSAG1 has been investigated as a candidate for recombinant vaccine development [28,29] and for immunodiagnosis [30][31][32]. SAG1, considered an antigen expressed only in tachyzoites [33], is expressed at equally high levels in tachyzoites and MNCs, and therefore would not account for an immunological differentiation of both stages. Conversely, in our experiment, the bradyzoite-specific NcSAG4 [34] is expressed in MNCs only, most likely by regulation on the transcriptional level. Similarly, SAG4 expression has been shown to be upregulated in intracellular parasites treated with artemisinin derivatives such as artemisone and GC007 [22]. However, these derivatives have a different mode of action, and treatments of parasites induced completely different alterations. Nevertheless, also artemisone and GC007 required in vitro treatments at a concentration of 5 µM over a period of over 20 days in order to act parasiticidal, similar to what has been observed for BKI-1294 [12]. Thus, upregulation of NcSAG4 expression could be a general reaction to adverse physiological conditions, similar to what has been postulated for bradyzoites and tissue cyst formation [35].
Besides SAG4, six other MNC-induced proteins contain signal peptides and thus may constitute surface antigens, or secretory proteins involved in modulating the parasite-host interface. Five other proteins have SRS-domains. These overexpressed proteins may constitute suitable candidates for immunological investigations in vivo and-if this hypothesis could be confirmed-for recombinant vaccine development.
Constitutive expression of NcSAG4 in N. caninum tachyzoites had been shown to confer protection against vertical transmission in a mouse model [36]. Therefore, it is conceivable that the excellent protective effects of BKI-1294 and related compounds against vertical transmission [12,[14][15][16] may be due to the induction of MNCs that express a set of diverse surface antigens, including NcSAG4. However, we currently do not know whether these antigens are expressed on the surface of newly formed zoites, or on the MNC surface. In any case, members of the SRS protein family are usually anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) molecule [37,38]. Besides NcSAG1 and NcSRS2 (also known as SRS29B and NcSRS29C, respectively), other SRS family members have been studied, including NcSRS67 that has no orthologue in T. gondii [39], and NcSRS57, which is expressed in both tachyzoites and bradyzoites [40,41]. However, according to our proteomics study, none of these appear to be overexpressed in MNCs.
Two additional studies described the development of similar multinucleated forms of the closely related T. gondii. Sugi et al. reported on another BKI named 1NM-PP1, which targeted T. gondii mitogen-activated protein kinase-like 1 (TgMAPKL-1), interfered in the cell cycle, and induced the formation enlarged multinucleated parasites. A mutation in a gatekeeper amino acid of TgMAPKL-1 led to a significant reduction in drug sensitivity and a reduction of enlarged multinucleated parasite formation [42]. In another study, a targeted deletion of the rab11a gene in T. gondii (TGME49_289680) also induced similar phenotypic changes [43]. The closest homologue in N. caninum, the unspecified protein NCLIV_041930, is expressed at similar levels in both tachyzoites and BKI-1294 induced MNCs (see Table S1).
Overall, we may consider that drug treatment, in the present case BKI-1294, does not only cause a shutdown of housekeeping gene expression and morphological alterations, but may also initiate "antigenic variation", albeit to a much lesser extent than in other protozoan parasites such as the apicomplexan Plasmodium sp. [44], the Excavata Trypanosoma sp. [45,46] and Giardia lamblia [47]. MNC formation may thus constitute a response strategy to ensure the survival in the presence of BKIs and perhaps other compounds. Here, further investigations are required, also with respect to the infective potential of these MNCs. It is noteworthy to mention that similar multinucleated stages were induced upon in vitro treatment of T. gondii with diclazuril [48]. Diclazuril is triazinone derivative that is effective against intracellular stages of Eimeria and Isospora spp. While the mechanism of this drug is not well understood, it was shown that diclazuril is targeting enzymes of the respiratory chain and other enzymes such as dihydrofolate reductase (DHFR) [49]. However, we propose that MNCs represent a stage designated as baryzoites (Greek βα υσ = massive, bulky, heavy, inert) as they represent a heavily enlarged parasitic stage that ensures survival of these parasites at increased drug concentrations during prolonged periods of time.