Introduction

Neosporosis is a protozoosis caused by Neospora caninum, which has a worldwide distribution (Dubey et al. 2007). N. caninum, which is classified in the phylum Apicomplexa (Dubey et al. 2002), can infect many species of animals, of which most importantly are cattle (Dubey and Schares 2011). N. caninum mainly parasitizes the muscle, liver, and central nervous system of the host leading to abortion, stillbirth, and fetal mummification of pregnant cows resulting in huge losses in the cattle industry (Reichel and Ellis 2008). Cell invasion by Apicomplexan protozoans is a parasite-driven process which displays substrate-dependent gliding (Soldati et al. 2004). Parasite gliding and microneme release are required for cell invasion (Keeley and Soldati 2004). However, the exact mechanism by which N. caninum invade host cells is still unknown.

The mitogen-activated protein kinase (MAPK) signaling pathway, including three main kinases p38 MAPK, extracellular signal-regulated protein kinase (ERK), and c-Jun N-terminal kinase (JNK), is essential in cell growth, proliferation, differentiation, and many other intracellular processes. p38 is a ubiquitously expressed enzyme that plays important roles in regulating cellular physiological processes (Martin-Blanco 2000). Possible MAP kinase sequences in Toxoplasma gondii and Plasmodium falciparum have been characterized (Lacey et al. 2007), but whether there are MAP kinase homologues in N. caninum remains to be identified. p38 MAPK has been shown to be necessarily involved in cell invasion by T. gondii (Valère et al. 2003). Studies show that p38 MAPK inhibitor SB203580 inhibits TgMAPK1 autophosphorylation and blocks intracellular T. gondii replication by direct effects on tachyzoites (Wei et al. 2002; Brumlik et al. 2004). Additionally, SB203580 inhibit sporozoite gliding motility, secretion of functional EtMICs, and cell invasion by Eimeria tenella (Bussière et al. 2015). However, whether blockade of the p38 MAPK pathway has any effect on N. caninum host cell invasion is not clear.

In the present study, we evaluated the effect of p38 MAPK blockade by the inhibitor SB203580 on tachyzoite motility, microneme protein (NcMIC2, 3, and 6) exocytosis, and host cell invasion by N. caninum.

Materials and methods

Antibodies

Mouse antiserum specific for NcMIC2, NcMIC3, and NcMIC6 and actin in N. caninum were prepared at our laboratory. p38 and phospho-p38 (Thr180/Tyr182) rabbit mAb were obtained from Cell Signaling Tech., Inc., MA, USA. Antibody for β-actin was obtained from Proteintech, PA, USA.

Cell culture

Vero cells and MDBK cells were cultured in DMEM (high glucose) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) and antibiotic–antimycotic reagents all from Life Technologies Co., CA, USA.

Parasite culture and purification

Tachyzoites of N. caninum-1 strain were stored at our laboratory. Vero cells were infected with tachyzoites of Nc-1 and cultured at 37 °C and 5 % CO2 for 3–5 days. After spontaneous cell rupture, tachyzoites and host cell debris were washed in cold DMEM without FBS and harvested by centrifugation at 850×g at 4 °C for 10 min. After centrifugation, the final pellet was resuspended in cold DMEM and passed through a 26-gauge needle (Millipore, Billerica, MA, USA). The obtained mixture was slowly layered on to a 40 % Percoll solution (GE Healthcare, USA) in DMEM without FBS and separated by centrifugation at 850×g in a horizontal centrifuge for 30 min. The fraction containing tachyzoites at the bottom of the tube was collected and resuspended in DMEM without FBS and centrifuged again at 850×g at 4 °C for 10 min. The purified tachyzoites in the pellet were resuspended in DMEM medium without FBS.

Tachyzoite motility assay

Tachyzoites (2 × 106) of Nc-1 strain were incubated in DMEM supplemented with 2 % FBS at 37 °C for 1 h in the presence of SB203580 (20 μM) or DMSO. For pretreatment, tachyzoites were pre-incubated with SB203580 (20 μM) or vehicle for 1 h. After washing three times with PBS, tachyzoites were allowed to glide. Tachyzoite motility was recorded for 30 s by videomicroscopy (Olympus BH-2, Japan) and assessed as described by Bumstead and Tomley (2000). Motile tachyzoites were observed and counted in five random fields of tachyzoites for each treatment.

Microneme protein secretion assay and immunoblot

Nc-1 tachyzoites (2 × 106) were incubated in DMEM supplemented with 5 % FBS in the presence of SB203580 (20 μM) or DMSO at 37 °C for 2 h, and tachyzoite pellets and supernatants were then collected by centrifugation at 850×g at 4 °C for 20 min. The tachyzoite pellet was resuspended in RIPA buffer supplemented with protease inhibitors (Sangon Biotech Co., Shanghai, China). After gentle sonication on ice for 30 s with 5-s pulses at 10-s intervals (Sonics and Materials Inc., USA), the tachyzoite pellet was lysed on ice. Tachyzoite lysates and culture supernatants were centrifuged at 10,000×g for 15 min at 4 °C, and both supernatants were collected. Supernatants were further mixed with loading buffer, boiled for 5 min, and electrophoresed on 12 % SDS-PAGE gels (Bio-Rad Laboratories, Inc., USA), then transferred to nitrocellulose membranes (Pall Life Sciences, USA). Membranes were blocked in 5 % skim milk (w/v) in TBST overnight at 4 °C. After washing, membranes were incubated with the primary antibody and then the corresponding secondary antibodies conjugated to horseradish peroxidase (1/2000; Proteintech, USA) in TBST at 37 °C for 2 and 1 h, respectively. Microneme proteins were detected by an enhanced chemiluminescence kit (Proteintech Group Inc., USA) using the ChemiScope series 5300 (Clinx Science Instruments Co., Ltd., Shanghai, China). Primary antibodies (1/200) were mouse anti-NcMIC2 (95–115 kDa), anti-NcMIC3 (38 kDa), and anti-NcMIC6 (25–40 kDa). The mouse antiserum against N. caninum (1/300, 45 kDa) was used as an internal control of parasite load. Microneme proteins in the culture supernatant and in the tachyzoite were quantified and compared by ImageJ software (NIH, USA).

Cell invasion by N. caninum

MDBK cells cultured in 24-well plates containing glass coverslips were infected with freshly purified Nc-1 tachyzoites in the presence of SB203580 (5–20 μM) or DMSO at a multiplicity of infection (MOI) of 10 at 37 °C in DMEM with 2 % FBS. The number of tachyzoites per 100 cells was detected 2 h after infection. The percentages of infected cells at 20 μM SB203580 were determined at 1 and 2 h post-infection respectively; for host cell pretreatment with SB203580, MDBK cells on coverslips were pre-incubated with or without SB203580 (20 μM) for 1 h, then coverslips were washed three times with PBS and infected with tachyzoites for 2 h. After infection, these coverslips were washed three times with PBS to remove the unentered tachyzoites. Cell monolayers were fixed with cold methyl alcohol for 10 min and stained with acridine orange (Life Technologies, USA) for 5 min. The percentage of infected cells or the number of tachyzoites per 100 cells was determined using a FV1000 confocal microscope (Olympus, Japan) by counting at ×1000 magnification. All experiments were done in triplicate, and at least 100 cells per sample were counted.

p38 MAPK activation analysis

Host cells, serum-starved overnight, were pre-incubated with or without 20 μM SB203580 (Selleck, USA) for 1 h, then washed with PBS and infected with or without Nc-1 tachyzoites at a MOI of 10 at 37 °C for 1 h. Cell pellets were collected and proteins were extracted. Protein concentration was determined using a BSA Protein Assay (Sangon Biotech, China), and then 50 μg proteins of each sample were separated by electrophoresis. p38 phosphorylation (Thr180/Tyr182) was detected by Western blot as described above. Beta-actin was used as the protein loading control.

Statistical analysis

Data were analyzed for statistical significance using Student’s t test and one-way ANOVA by SPSS 19.0 software (InStat version 3.0, GraphPad, La Jolla, CA). Results are expressed as the mean ± standard errors. Differences were considered statistically significant when P values were <0.05.

Results

Putative p38 MAPK homologues in N. caninum

To search for putative p38 MAPK homologues in N. caninum, the mouse p38α MAPK protein (NP_001161980; Mapk14, Mus musculus) was blasted on the N. caninum database (www.toxodb.org). The first five kinase homologues with the lowest e values are presented in Table 1. All the five homologue proteins belong to the MAP kinase family and the cyclin-dependent like kinases (CDK) family proteins sharing 34–44 % identity with the mouse p38 MAPK. Sequence analysis of these kinase homologues identified the kinase catalytic domain motifs with the high conservation. As shown in Table 2, the amino acids K53 of the motif VAXK (subdomain II), D168 of the motif DFGLAR (subdomain VII), and T180 of the motif T180XY182TXXYXAPE (subdomain VIII) of the mammalian p38α MAPK were found in all five homologue kinase proteins. T180 and Y182 of the motif TXYTXXYXAPE, which are the main phosphorylation sites of p38 MAPK, were both present in kinase homologues NCLIV_032840, NCLIV_015030, and NCLIV_056080. The results indicated that SB203580 might act directly on N. caninum.

Table 1 The first five homologue proteins of p38α MAPK in N. caninum
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Table 2 Alignment of N. caninum MAPK homologue proteins to the mouse p38α MAPK
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SB203580 reduced N. caninum tachyzoite motility

The results showed that SB203580 caused a marked reduction of 55.3 % in the percentage of motile tachyzoites, which was recovered when the inhibitor was removed (Fig. 1), indicating that SB203580 had a direct inhibitory effect on tachyzoite motility of N. caninum.

Fig. 1
figure 1

Impact of SB203580 on tachyzoite motility of N. caninum. For SB203580 treatment, N. caninum tachyzoites were incubated with SB203580 (20 μM) or DMSO (Ctr) at 37 °C for 1 h; for pretreatment, the inhibitor was then washed off and Nc-1 tachyzoites were allowed to glide for 1 h. Tachyzoite motility was examined for 30 s. Motile tachyzoites for five random fields per treatment were counted. Data represent the mean of three independent experiments ± SEM. Differences are considered statistically significant compared to Ctr when P was <0.05

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SB203580 inhibited the exocytosis of N. caninum microneme proteins

Microneme protein secretion was induced by FBS and detected by Western blotting using specific antibodies against corresponding NcMICs, respectively. Results showed that the secretions of NcMIC2, 3, and 6 were all significantly reduced when treated with SB203580, as shown in Fig. 2, indicating that SB203580 inhibited microneme protein exocytosis by N. caninum tachyzoites.

Fig. 2
figure 2

Impact of SB203580 on exocytosis of microneme proteins by N. caninum. Tachyzoites were incubated in DMEM with 5 % FBS in the presence of 20 μM SB203580 or not for 2 h at 37 °C and then centrifuged to gather tachyzoites and supernatants. Tachyzoite pellets were then sonicated and lysed. The tachyzoite lysate and medium supernatant were subject to immunoblot to examine the microneme secretion using antiserum specific for NcMIC2, 3, and 6. An actin in N. caninum was used as loading control of parasites. The intensity of the protein bands in the Western blot is quantified by ImageJ (NIH) software. The exocytosis of microneme proteins is represented by comparing the NcMIC content in the culture medium to that in the tachyzoite lysate. Results are representative of three independent experiments

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SB203580 effectively inhibited MDBK cell invasion by N. caninum

MDBK cells were inoculated with N. caninum tachyzoites either in the presence of SB203580 or in its absence. The inhibition of cell invasion by SB203580 was dose-dependent. SB203580 at concentrations of 5, 10, and 20 μM caused decreases of 20.4, 39.8, and 63.5 %, respectively, in the numbers of tachyzoites per 100 cells 2 h post-infection (Fig. 3a). With 20 μM, SB203580 reduced the percentage of infected cells by 54.8 and 57.3 % at 1 and 2 h, respectively (Fig. 3b). Those data showed that SB203580 could effectively inhibit host cell invasion by N. caninum.

Fig. 3
figure 3

The p38 MAPK inhibitor SB203580 inhibits cell invasion by N. caninum. a Effect of SB203580 on MDBK cell invasion by N. caninum tachyzoites in the presence of SB203580 with different concentrations 2 h post-infection (0–20 μM, 0 μM was treated with same amount of DMSO). Cell invasion is expressed as the number of tachyzoites per 100 cells. b Effect of SB203580 on MDBK cell invasion by N. caninum tachyzoites in the presence of 20 μM SB203580 or DMSO (Ctr) at 1 and 2 h, respectively. Cell invasion is expressed as the percentage of infected cells. Results are representative of three independent experiments. Data are expressed as the mean ± SEM. Differences are considered statistically significant compared to Ctr when P was <0.05

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SB203580 inhibited cell invasion by N. caninum working both on parasites and host cells

MDBK cells were pre-incubated with SB203580 or DMSO and rinsed with PBS before infection with N. caninum tachyzoites. The percentage of infected cells at 2 h was reduced by 32.8 % as shown in Fig. 4a. Furthermore, in host cells infected with N. caninum, the p38 MAPK was phosphorylated, and SB203580 effectively inhibited host p38 activation induced by N. caninum infection (Fig. 4b). These results indicated that the host cell p38 MAPK is also involved in the cell invasion by N. caninum.

Fig. 4
figure 4

The host p38 MAPK also plays some roles on cell invasion by N. caninum. a MDBK cells were pretreated with SB203580 (20 μM) or DMSO (Ctr) for 1 h, then washed off the inhibitor before infection with N. caninum tachyzoites for 2 h. Cell invasion is represented as the percentage of infected cells. b p38 MAPK activation analysis of host cells. SB203580- or DMSO-pretreated MDBK cells were infected with or without N. caninum tachyzoites at a MOI of 10 for 1 h. p38 phosphorylation was determined by Western blot. β-actin was used as the loading control of proteins. Results are representative of three independent experiments. Data are expressed as the mean ± SEM. Differences are considered statistically significant compared to Ctr when P was <0.05

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Discussion

Like many other Apicomplexan protozoans, host cell invasion by N. caninum is a subtle and complicated process including parasite gliding, attachment to host cells, formation of the moving junction, and a series of complex processes accompanied by sequential secretion of a series of proteins from distinct secretory compartments located at the apical end of the parasite, including micronemes, rhoptries, and dense granules (Sibley 2004). Nevertheless, the precise mechanism of cell invasion by N. caninum which is regulated by various signal transduction pathways is not entirely defined. In the current study, the impact of blocking the p38 MAPK pathway by SB203580, a competitive inhibitor targeting ATP binding sites, on host cell invasion by N. caninum was investigated.

First, we discovered the presence of putative p38 MAPK homologues in N. caninum with eukaryotic protein kinase catalytic domain motifs, which might represent drug development targets for anti-parasitic agents. Although a recent study shows that SB203580 did not completely block TgMAPK1 (Brown et al. 2014), whether SB203580 could act on NcMAPK1 or other MAPK homologues in N. caninum is still not clear. The homologue of TgMAPK1 in N. caninum might be NCLIV_056080, which presents 65 % amino acid sequence identity with TgMAPK1 and its threonine, aspartic acid, and tyrosine (TDY) activation loop is consistent with known apicomplexa MAPK sequences. T180 and Y182, phosphorylation by MAPK kinases leading to the activation of p38 MAPK, together with K53 and D168 (all numbered in mouse p38 MAPK) are necessary for catalytic activity. These motifs are highly conserved between mouse p38 MAPK and parasite kinase homologues, especially the homologue proteins NCLIV_032840, NCLIV_015030, and NCLIV_056080 which probably are the targets for SB203580.

Next, our results showed that SB203580 caused significant reductions both in the percentage of motile tachyzoites and in the extracellular secretion of NcMIC2, 3, and 6, which indicate from two aspects that SB203580 had direct effects on N. caninum. Host cell recognition, adhesion, and invasion depend on parasite gliding and are accompanied by exocytosis of microneme proteins (MICs). MIC discharge occurs early in cell invasion. In apicomplexans, host cell invasion has been demonstrated to be closely associated with MIC secretion (Dowse and Soldati 2004), especially thrombospondin-related anonymous protein (TRAPs) shown to be necessary for parasite motility and invasion by T. gondii (Huynh and Carruthers 2006) and P. falciparum (Morahan et al. 2009). Micronemes from N. caninum tachyzoites contain many MICs, most of which possess recognizable, vertebrate-derived adhesive domains critical for interactions between parasites and hosts (Carruthers and Tomley 2008). In this study, we examined NcMIC2, 3, and 6. NcMIC2 contains an integrin-like I/A-domain and five thrombospondin type I-like repeats. NcMIC3 contains a lectin-like domain and four epidermal growth factor (EGF) repeats (Yang et al. 2015). They both have been demonstrated to have host cell binding properties and important for cell invasion by N. caninum (Naguleswaran et al. 2001; Pereira et al. 2011). NcMIC6 possesses three EGF-like domains (Li et al. 2015), but its precise function has not been identified. The reduction in the secretion of NcMIC2, 3, and 6 might hinder cell adhesion and invasion by N. caninum and subsequently the proliferation within host cells.

Subsequently, the impact of SB203580 on parasite burden during the invasion stage by N. caninum was examined in MDBK cells. Our results showed that SB203580 at concentration of 20 μM, which is close to the highest IC50 (Bussière et al. 2015), exhibited no toxicity to host cells but caused decreases of 63.5 % in the number of tachyzoites per 100 cells 2 h post-infection and 54.8 and 57.3 % in the percentage of infected cells at 1 and 2 h, respectively. Furthermore, when only host cells were pretreated with SB203580, p38 MAPK activation induced by N. caninum was effectively inhibited and the percentage of infected cells was reduced by 32.8 %, indicating that except for parasite MAPKs, host p38 MAPKs also participated in cell invasion by N. caninum. These data distinctly demonstrate that SB203580 can inhibit host cell invasion by N. caninum. The precise mechanism of cell invasion inhibition during host p38 MAPK blockage remains to be identified.

Protein kinases have been confirmed to be significant in regulating host cell invasion, survival, and parasite proliferation (Kim 2004; Sibley 2013; Wei et al. 2013). It has been reported that activation of the PI3 Kinase/Akt pathway is essential for intracellular proliferation of T. gondii (Zhou et al. 2013). T. gondii infection activates EGFR-AKT signaling, and furthermore AG1478 (a EGF receptor inhibitor) and AKT inhibitor IV can impair parasite survival in host cells (Muniz-Feliciano et al. 2013). Isoflavone analogs, targeting tyrosine kinase of EGFR, inhibit development of Sarcocystis neurona, Cryptosporidium parvum, and N. caninum (Gargala et al. 2005). Serine protease inhibitors block invasion of host cells by T. gondii (Alam 2014) and P. falciparum (Conseil et al. 1999). JNK inhibitor SP600125 reduces neuronal cell death in experimental cerebral malaria (Anand et al. 2013). p38 inhibitors block replication of P. falciparum and T. gondii (Brumlik et al. 2011). In other Apicomplexa infections such as T. gondii, kinase inhibitors targeting MAP kinases have been shown to reduce host cell invasion (Robert-Gangneux et al. 2000). Recent studies suggest that JNK inhibitor SP600125 inhibits cell invasion by E. tenella; p38 MAPK inhibitor SB203580 significantly decreases parasite motility and micronemal protein secretion which cause a serious decrease of cell invasion by E. tenella, with 25 μM decreasing the percentage of infected cells by 91 and 85 % in MDBK and m-ICcL2, respectively (Bussière et al. 2015). However, the kinases and signal transduction pathways involved in host cell invasion are poorly understood in N. caninum.

In summary, in this study, we report the novel finding that the p38 MAPK inhibitor SB203580 affects both N. caninum and host cells to inhibit tachyzoite motility, MIC exocytosis, and ultimately cell invasion. Taken together, current information indicates that SB203580 could be useful for chemotherapy of neosporosis. However, further studies are needed to identify the MAP kinase activity and precise function of p38 MAPK in cell invasion and infection by N. caninum.