Role of GRA41 in Neospora caninum pathogenicity: insights into tachyzoite egress and microneme secretion

doi:10.21203/rs.3.rs-3910133/v1

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Role of GRA41 in Neospora caninum pathogenicity: insights into tachyzoite egress and microneme secretion

https://doi.org/10.21203/rs.3.rs-3910133/v1

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Background

Egress represents a crucial process employed by Neospora caninum in the establishment of infection. Dense granule proteins (GRAs), discharged by the dense granule, an essential secretory organelle of Neospora caninum, significantly contribute to the modification of parasitophorous vacuole, maintenance of morphology, and regulation of host cells. However, the precise involvement of these proteins in the egress process of tachyzoites remains inadequately characterized.

Methods

Comprehensive searches and comparative analyses were conducted of the homologous gene with dense granule protein 41 of Toxoplasma gondii, utilizing the NCBI and ToxoDB databases. Subsequently, we performed online bioinformatics analysis. Additionally, for ascertaining subcellular localization, we created an endogenously labeled strain expressing NcGRA41-3xHA. Employing CRISPR/Cas9 gene editing technology, we constructed the NcGRA41 knockout strain (Δncgra41) and NcGRA41 complementary strain (comΔncgra41) to analyze its phenotypes. To further elucidate the function role of NcGRA41, we performed a micronemes secretion assay and assessed the transcription levels of relevant factors during tachyzoites egress through RT-qPCR.

Results

NcGRA41 exhibited extracellular localization within dense granules and intracellular distribution in parasitic vacuoles. Deletion of NcGRA41 had no discernible impact on the invasion and proliferation of tachyzoites, but, markedly reducing the capacity for egress and pathogenicity in mice. The complementary strain recovered the phenotypic characteristic of Nc1 parasites. Further investigation revealed that the absence of NcGRA41 led to a reduction in gliding motility and the transcription level of subtilisin-like protein (SUB1). The microneme secretion assay demonstrated a significant decrease in the secretion level of NcMIC1, accompanied by reduced expression levels of NcMIC1, NcMIC4, and NcMIC8. These findings collectively contributed to the ultimate decrease in egress.

Conclusions

The identification of a novel Neospora caninum dense granule protein, designated NcGRA41, has been achieved. NcGRA41 is implicated in influencing the pathogenicity of N. caninum by modulating tachyzoites egress through the secretion of micronemes.

Neospora caninum

Dense granule protein 41

Egress

Microneme secretion

pathogenicity

Neospora caninum is an obligate intracellular parasite belonging to Apicomplexa, considered an important agent of bovine abortion, capable of infecting cattle, sheep, dogs, and other animals [1]. Although this protozoan does not infect humans, it has inflicting significant economic losses on the animal husbandry industry, particularly in cattle [2, 3]. The lytic cycle of N. caninum encompassed invasion, intracellular proliferation, and egress. Considered as a pivotal step in establishing infection, egress has been extensively studied in the model parasite Toxoplasma gondii, which activates microneme exocytosis and the actomyosin system through a series of molecular mechanisms, ultimately triggering the egress of tachyzoites from the host cell [4, 5, 6, 7]. When these signals are sensed, micronemes release perforin, lipase, protease, and adhesin, playing crucial roles in egress, gliding, and invasion [8]. Although some signal pathways related to this process are conserved between T. gondii and N. caninum, the key molecules involved in egress are sparsely investigated in N. caninum [9].

Dense granule (GRA) is one of three specific secretory organelles of Apicomplexan, including N. caninum [10]. Proteins previously identified secreted by dense granule played vital roles in modifying vacuoles, maintaining the morphology of the intravacuolar network (IVN), transporting small molecules, regulating host cells, cyst development, and tachyzoites egress [11, 12, 13, 14, 15, 16]. Nevertheless, the functions of GRA proteins have been rarely determined in N. caninum, especially the role of GRA proteins in the egress process, which is poorly understood [17]. Among the currently reported five T. gondii GRA proteins involved in the parasite egress, TgGRA41 plays a critical role in calcium homeostasis and the timing of natural non-induced egress [18, 19, 20, 21]. We hypothesized that the homologous gene of Tggra41 in N. caninum could contribute to a similar function.

In this study, we identified a novel N. caninum dense granule protein, NcGRA41, and demonstrated that the absence of NcGRA41 decreases the motility of parasites, the transcription level of SUB1, as well as the secretion of some MICs, ultimately resulting in the reduced egress ability of parasites. Together, this study initially confirms the role of NcGRA41 in the egress process of N. caninum.

Parasites and cell cultures

N. caninum tachyzoites were cultured and passaged in Human Foreskin Fibroblast (HFF) and African Green Monkey kidney cells (vero) lines. The cells and parasites were maintained in a humidified incubator at 37°C with a 5% CO₂ concentration. The standard growth medium used was Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 25 mM glucose and 4 mM glutamine.

Generation of transgenic strains

The primer sequences are available in Table S1.

For the generation of NcGRA41-3xHA parasites, sgRNA was designed from the ncgra41 stop codon downstream, approximately 200-300bp sequence, using the ToxoDB database. The sgRNA sequence was incorporated to U6 downstream and AMP upstream primers. Subsequently, the Cas9 skeleton, U6 promotor and Amp fragments were amplified from the previously constructed pNcCRISPR/Cas9 plasmid in our lab. These fragments were then ligated by seamless cloning to form a new pNcCRISPR/Cas9 plasmid, named pNcCRISPR/Cas9::sgNcGRA41. The 5′ and 3′ flanks of the ncgra41 stop codon, front 1000bp, and sgRNA behind 1000bp sequences were amplified from the DNA of Nc1 parasites and inserted into the backbone of the pLIC-GRA41-DHFR-3×HA plasmid using seamless cloning. The 5′ flank-DHFR-3×HA-3′ flank fragment from the newly constructed pLIC-GRA41-DHFR-3×HA plasmid and pNcCRISPR/Cas9::sgNcGRA41 plasmid were co-transfected into Nc1 parasites and screened with pyrimethamine. Clones were verified through western blot (WB) and immunofluorescence assay (IFA).

To construct the NcGRA41 deficient strain (Δncgra41), 5′ and 3′ flank fragment of NcGRA41 gene before and behind 1000bp sequence were amplified from the genomic DNA of Nc1 parasites. DHFR and pTCR backbone were amplified from the previously generated pTCR-DHFR plasmid. The fragments were linked and sequenced to form a new pTCR-DHFR-NcGRA41 plasmid. The 5′ flank-DHFR-3′ flank fragment was then amplified from this plasmid. The pCRISPR/CAS9::sgΔncgra41 was constructed using a similar method as described above, with the sgRNA selected from the NcGRA41 gene. The linearized 5′ flank-DHFR-3′ flank fragment and pNcCRISPR/Cas9::sgΔncgra41 plasmid were co-transfected into Nc1 parasites and screened with pyrimethamine. Clones were verified through PCR.

For the generation of NcGRA41 complemented parasites (comΔncgra41), sgRNA was selected from the NcUPRT locus, and the pCRISPR/CAS9::sgNcUPRT was constructed accordingly, with sgRNA selected from the NcUPRT locus. The NcUPRT gene, before and behind 1000bp sequence, and the NcGRA41 gene were amplified from the DNA of Nc1 parasites. These fragments were then inserted into the pNcUPRT::DHFR backbone vector by homologous recombination to construct the pNcUPRT::DHFR-NcGRA41 plasmid. Finally, the pNcCRISPR/Cas9::sgNcUPRT plasmid and linearized fragment were co-transfected into Δncgra41 strain, and the complementary parasites were selected using fluorodeoxyribose (FUDR) and confirmed through PCR and IFA.

Western blot

Freshly isolated parasites were harvested and purified through a 5 µm filter, followed by centrifugation at 1400×g for 8 min, and washing in PBS. Purified tachyzoites were lysed in radio immunoprecipitation assay lysis buffer (RIPA) containing a cocktail of protease inhibitors. The lysates were then separated on 12.5% (w/v) SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% (w/v) powdered milk (BD Difco, USA) and probed with a 1:500 dilution of mouse anti-HA antibody (Sigma-Aldrich) for 1 h. Mouse anti-NcActin antibody (National Animal Protozoa Laboratory, China Agricultural University) was used as a loading control for N. caninum F-actin subunit beta. After washing with PBST (PBS with 1% Tween-20), the membranes were incubated with an HRP-conjugated goat anti-mouse IgG (Sigma, USA) at 1:5 000 dilution for 1 h. Following three washes with PBST, visualization was performed using Plus ECL (CoWin Biotech Co., Ltd, China).

Immunofluorescence assay

Tachyzoites (1×10⁴) were seeded onto HFFs previously prepared on glass coverslips in 12-well plates and incubated at 37℃ with 5% CO₂ for 30 h. and then fixed for 30 in 4% formaldehyde for 30 min. Subsequently, cells were permeabilized with 0.25% Triton X-100 for 15 min, followed by blocking with 3% bovine serum albumin (BSA) for 1 h. The cells were incubated with a mouse anti-HA monoclonal antibody (1:500, Sigma-Aldrich), rabbit anti-NcGRA1 polyclonal antibody (1:200), and rabbit anti-NcSRS2 polyclonal antibody (1:500) at 37 ℃ for 1 h. Following incubation, parasitess were treated with FITC-conjugated goat-anti mouse IgG (H + L) and Cy3-conjugated goat-anti rabbit IgG (H + L) (Sigma, USA). Hoechst33258 (1:100, Sigma, USA) was used for nuclear DNA staining. Images were captured with a Leica confocal microscope system (Leica, TCS SP52, Germany). Antibodies against NcGRA1 and NcSRS2 were provided by the National Animal Protozoa Laboratory, China Agricultural University. To analyze NcGRA41 localization in extracellular parasites, filtered parasites were allowed to adhere to coverslips precoated with poly-lysine at 4℃ overnight. The IFA process and image collection followed the procedures described above.

Plaque assay

Plaque assay was performed using HFFs cultured in 12-well cell culture plates. The HFFs monolayer, reaching 80–90% confluency, was infected with 200 freshly isolated parasites per well and then incubated in a 37°C incubator with 5% CO₂ for 7 days. After incubation, cells were washed twice with PBS, fixed with 4% paraformaldehyde, and stained with 2% crystal violet solution for 2–4 h. Plaque areas were measured as described previously, and data were compiled from three independent experiments [22].

Invasion assay

Invasion assay was performed by inoculating freshly isolated parasites (1×10⁶) on HFFs in12-well cell culture plates. After invasion for 1 hour at 37°C, extracellular parasites were removed by washing with PBS. Following 30 h of incubation, infected cells were fixed with 4% paraformaldehyde. Parasites were then stained with a rabbit anti-NcSRS2 polyclonal antibody (1:500), and host cell nucleus were stained with Hoechst33258 (1:100, Sigma, USA) following the IFA protocol. For each experiment, a minimum of 100 parasites were counted from 10 fields per treatment. Invasion efficiency was calculated as the ratio of the numbers of vacuoles to the numbers of host nuclei. Results represent the mean ± standard deviation of three independent biological replicate experiments.

Proliferation assay

A total of 1×10⁶ freshly tachyzoites were inoculated into HFF cells cultured in 12-well cell culture plates. After 1 hour invasion, the cells were washed with PBS three times, and fresh medium was added for continuously cultivation for 30 h. Infected HFFs were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100. The parasites were labeled with a rabbit anti-NcSRS2 polyclonal antibody (1:500), and the nucleus was stained with Hoechst33258. At least 100 vacuoles were quantified per strain per experiment, and the number of tachyzoites contained in each vacuole was counted separately (e.g., 2, 4, 8, 16 tachyzoites). Three independent experiments were conducted.

Egress assay

HFFs monolayers on 12-well coverslip were infected with 1×10⁵ parasites for 1 hour at 37°C. The medium containing uninvaded parasites was removed, and the cells were washed with PBS. Fresh 2% FBS DHEM medium was added and the cells were allowed to grow for 30–33 h until large vacuoles were visualized, but parasites were not released, as observed under an inverted microscope. For induction of egress, host cells were incubated for 3–5 min with PBS containing 1 µM A23187 or 5% absolute ethyl alcohol. This process was carried out in the dark. Following this, cells were fixed with 4% paraformaldehyde, and the subsequent IFA assay was conducted, connecting with invasion protocol. A minimum of 100 parasites were counted from randomly selected fields. The efficiency of egress was calculated as the ratio of vacuoles with released parasites to the total counted number of vacuoles. Three independent experiments were performed.

Gliding assay

Glass coverslips were coated overnight at 4°C with 20% FBS dissolved in PBS. Freshly lysed and filtered tachyzoites were resuspended in Ringer buffer (composition: NaCl 9.1 g, KCl 0.23 g, CaCl₂·2H₂O 0.29 g, MgCl₂ ·6H₂O 0.2 g, NaH₂PO₄·2H₂O 0.468 g, HEPEs 2.4 g, Glucose 1.8 g, dissolved in 900 mL distilled water). Subsequently, 250 µL of this suspension was added to the coated slides and incubated for 4–5 min on ice. Ringer buffer containing 2 µM A23187 was then added, followed by incubation for 20 min at 37℃. The slides were fixed with 4% paraformaldehyde. Trails left by gliding parasites were visualized by staining with rabbit anti-NcSRS2 antibody (1:500). A total of 100 parasites were enumerated per strain/treatment, and the numbers of parasites trails was recoded to calculate the proportion of gliding tachyzoites. Trail lengths were measured using ImageJ. Three independent experiments were performed.

Parasite virulence assay

To assess the virulence of the transgenic strain compared to the Nc1 strain, groups of 5 female BALB/c mice aged between 7–8 weeks were intraperitoneally (i. p.) infected with 8×10⁶ tachyzoites post-adaptation. Mice were monitored daily over a 60-day period for clinical symptoms and survival time post-infection. Data were collected from three independent experiments. Throughout the entire experiment, mice were provided with an ample food and water, along with appropriate environmental conditions to ensure their well-being.

Quantitative real-time PCR

A total of 1×10⁷ tachyzoites collected at 46–48 h post-invasion were used for total RNA extraction following the manufacturer’s instructions with TRIZOL® reagent (Invitrogen). The concentration of RNA was measured using a NanoDrop 2000c spectrophotometer (Thermo Scientific). Equal amounts of total RNA were then synthesized into cDNA utilizing a First-Strand cDNA Synthesis kit (Invitrogen). ABgene Absolute qPCR SYBR Green Master Mix (ABgene, Surrey, UK) with ROX dye was used for all PCR protocols. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) reactions were conducted in triplicate in 96-well plates on an ABI Prism®7900HT real-time PCR machine. The thermal cycling conditions consisted of one cycle at 50°C for 2 min for enzyme activation, followed by one cycle at 95°C for 15 min for enzyme activation. Subsequently, forty cycles were run at 95°C for 15 sec for denaturation and 60°C for 1 min for the anneal/extension step. The threshold cycle number (Ct) of the target gene was calculated, with NcActin serving as the reference gene. Delta–delta Ct values of the genes were presented as relative fold induction.

Microneme secretion assay

The microneme secretion assay was conducted following a previously established protocol [23]. In brief, 1×10⁸ purified tachyzoites from both Nc1 and Δncgra41 strains were suspended in 1 mL DMEM containing 1 µM A23187. The mixture was incubated at 37°C in a water bath for 30 min. Subsequently, the mixture was centrifuged at 1300×g for 15 min at 4°C to separate the supernatant and precipitation. The precipitation was then combined with 98 µL RIPA and 2 µL Cocktail, followed by a 30 min incubation on ice. The supernatants and lysate were processed using SDS-PAGE followed by a western blot protocol to detect the secretion and expression of NcMICs. The primary antibodies used included mouse anti-NcActin monoclonal antibody (1:4 000), mouse anti-NcMIC1 polyclonal antibody (1:300), mouse anti-NcMIC4 polyclonal antibody (1:300), and mouse anti-NcMIC8 polyclonal antibody (1:1 000). The secondary antibody used was an HRP labeled goat anti-mouse lgG monoclonal antibody (1:5 000).

Statistical analysis

The statistical analysis was conducted using GraphPad Prism 5. Log-rank (Mantel-Cox) Test was used for data analysis. The results were presented as mean ± SD, and values with P < 0.05 and P < 0.01 were considered statistically significant.

Identification of the N. caninum dense granule protein NcGRA41

Given that Tggra41, related to the parasites’ egress process, has been previously reported in T. gondii (accession number TGGT1_069070, ToxoDB v7.2), we aimed to explore its homologous gene in N. caninum. However, we encountered difficulty retrieving the published Tggra41accession number from the updated ToxoDB (http://toxodb.org/toxo/). Subsequently, we searched for the Tggra41 sequence corresponding to the published protein sequence in the NCBI database. The identified sequence was subsequently compared in the ToxoDB database, and we eventually determined the position of the homologous gene sequence of Neospora (FR823391: 5147741–5148296). This protein is predicted to have a size of 20.248 kDa and shares 45% amino acid similarity with Tggra41 (Fig. 1a). The predicted protein possesses a signal peptide at amino acids 1–30 and lacks a transmembrane domain. The secondary structure analysis revealed that the sequence contains an alpha helix (42.39%), an extended strand (8.15%), a beta turn stack (4.89%), and a random coil (44.57%) (Fig. 1b). The three-dimensional (3D) structures further determined that this protein is similar and closely related to TgGRA41 (Fig. 1c).

To confirm the subcellular localization of FR823391: 5147741–5148296, we generated a parasite strain expressing this protein tagged at its C terminus with an HA epitope, as confirmed by western blot analysis (Fig. 1d). Subsequently, the IFA was carried out to determine the localization of this protein using anti-HA antibody. The results showed that FR823391: 5147741–5148296 is distributed intracellularly in the parasitophorous vacuole and extracellularly in dense granules, co-localizing with the dense granule marker GRA1 (Fig. 1e). These findings suggest that the encoded protein is a dense granule protein, and hence, we designated it as NcGRA41.

NcGRA41-deficient parasites delay the egress of tachyzoites and reduce virulence

Given the homology between Ncgar41 and Tggra41, we aimed to further explore the function and mechanism of Ncgar41 in N. caninum. We initially disrupted the Ncgra41 gene by replacing the entire locus with the selectable marker DHFR in the parental Nc1 strain. The successful generation of knockout parasites, named Δncgra41, was confirmed through PCR (Fig.S1 b). Additionally, we reintroduced the Ncgra41 gene at the NcUPRT site to verify the function of NcGRA41. The successful generation of the complemented strain, named comΔncgra41, was confirmed through both PCR and IFA (Fig.S1 d, e ).

We utilized those gene-edited strains along with the Nc1 parental strain to compare their ability for plaque formation over 7 days. Plaque assay revealed that the plaque size of the strain lacking NcGRA41 was smaller than that of Nc1 and comΔncgra41, implying a role for NcGRA41 in one or several steps of the parasite’s lytic cycle (Fig. 2a, b). Consequently, we conducted invasion, proliferation, and egress assays on the three strains, respectively. Although there were no statistically significant differences in invasion (Fig. 2c) and replication among these strains (Fig. 2d), the proportion of egressed Δncgra41 strain induced by 1 µM calcium ionophore A23187 was significantly lower than that of Nc1 and comΔncgra41 strains. This explained that the smaller plaque formation by Δncgra41 was due to a delay in egress (Fig. 2e).

Furthermore, we investigated the virulence between the Δncgra41 strain and Nc1. We administered a dose of 8×10⁶ tachyzoites intraperitoneally to BALB/c mice. By 5 days post-infection, mice infected with the parental strain Nc1 displayed depression and rough coat, with eighty percent of mice succumbing within 10 days. In comparison, the Δncgra41 strain exhibited an attenuation of virulence. Mice did not show clinical symptoms until 45 days, and all died within 52 days (Fig. 2f). Our findings suggest that NcGRA41 contributes to Neospora virulence by regulating tachyzoites egress.

Lacking NcGRA41 affects the gliding motility of N. caninum

The motility ability of parasites is crucial for the egress process. To understand why the deficiency of NcGRA41 affects tachyzoites egress, we initially performed a gliding motility assay, a commonly used in vitro method that assesses trail formation on FBS-coated coverslips. The gliding behavior was analyzed by measuring trail length and recording the proportion of trails. Fewer trails were detected for the Δncgra41 strains compared to Nc1 or comgra41 (Fig. 3a, b). Furthermore, there was a significant quantitative difference in gliding distance between Δncgra41 and Nc1. The Nc1 and comgra41 strain showed longer gliding distance than Δncgra41 (Fig. 3c). These results indicate that the absence of NcGRA41 weakened the gliding motility of Neospora.

Deletion of NcGRA41 affects secretion and expression of some NcMICs

Numerous studies have reported that microneme exocytosis plays key roles in the egress and motility of parasites. For further exploration of whether NcGRA41 affects the secretion or expression level of micronemes, we conducted a microneme secretion assay. After stimulating freshly collected intracellular tachyzoites with 1 µM A23187 for 30 min, a western blot was performed using the pellet and supernatant. The results showed that NcMIC1, NcMIC4, and NcMIC8 in the Δncgra41 strain could still be secreted out of the cell. However, the amount of NcMIC1 secreted into the supernatant was significantly less than that of Nc1, while the pellet of NcMIC1, NcMIC4, and NcMIC8 were also reduced compared with Nc1 (Fig. 4a-b). These results implied that the deletion of NcGRA41 caused a decrease in NcMIC1 secretion and expression of NcMIC1, NcMIC4, and NcMIC8.

NcGRA41 deletion affects the transcription level of SUB1 genes

The maturation and secretion of microneme proteins are regulated by various genes, including TgSUB1 responsible for the cleavage of MICs and protein kinase A of T. gondii considered a negative regulator of MICs secretion, among others. Consistent with the molecules related to the microneme exocytosis process in Toxoplasma and Plasmodium, we identified corresponding homologous genes in Neospora, including pka, pkg, cdpk3, cdc50, gpcr, sub1, ugo and doc2 genes. Subsequently, RT-qPCR was performed to detect whether the transcription level of these molecules in the NcGRA41 deletion strain were altered. The results revealed a significant reduction in the transcription level of SUB1 in the Δncgra41 strain among the tested genes (Fig. 4c), which may contribute to the decreased secretion and expression of NcMIC1.

In light of the critical role of the egress process in establishing protozoan Apicomplexan infections, there is a growing interest in studying the molecules involved in this process. In recent years, several T. gondii GRAs, such as TgGRA22, TgGRA41, and TgLCAT, have been reported to regulate the egress [18, 19, 20]. In comparison to T. gondii, there is limited knowledge about the identification and functional analysis of GRAs in N. caninum. Therefore, we dedicated particular attention to investigating the potential impact of T. gondii homologous GRAs on the Neospora egress process, with a primary focus on GRA41 in this study.

Bioinformatics analysis of the newly identified NcGRA41 revealed that it contains six alpha helices and one signal peptide, consistent with the characteristics of most dense granule proteins [24]. These two structures are essential for the subcellular localization and biological functions of many GRAs. NcGRA41 is located intracellularly in the parasitic vacuole and extracellularly in dense granules. We hypothesized that it is also related to the egress process and further explored its functional mechanism.

The NcGRA41 knockout strain exhibited a weakened ability to induce egress and pathogenicity in mice. However, the mechanism by which Δncgra41 strain affects escape ability differs from that of Δtggra41 strain. TgGRA41 leads to early egress of parasites, which would account for a majority of the decrease in plaque size [20]. Conversely, NcGRA41 resulted in delay escape induced by A23187 in this study. This difference is possibly attributed to variations in the types of strains. Furthermore, it is worth noting that TgGRA41 may regulate Ca²⁺ homeostasis, and whether NcGRA41 is related to the regulation of Neospora Ca²⁺ remains to be further confirmed. This could be conducted by using a calcium probe detect calcium levels changes of N. caninum under the loss of NcGRA41. Moreover, genetically encoded Ca²⁺ indicators inside parasites could be expressed by transgenic approaches, which can also be used to measure Ca²⁺ during the lytic cycle of the parasites.

To further explore the cause of Δncgra41 egress delay, we investigated transcription levels of some important genes involved in the egress process of T. gondii and Plasmodium. Among them, CDC50.1 is considered as a partner molecule of the GC structure of T. gondii, playing an indispensable function upstream of the egress signal cascade [21]. PKA, as a negative regulator [25], PKG, as an important target molecule of cGMP [26, 27], and CDPK3, as a calcium-dependent protein kinase, all regulate microneme exocytosis during the egress process [7]. TgSUB1 is involved in the maturation and cleavage of the microneme proteins, affecting parasites’ invasion, adhesion, and gliding motility. Parasites lacking TgSUB1 have a virulence defect leading to a delayed time of death in the mouse model [28]. Additional evidence suggests that a decrease in the mature of SUB1 cause the accumulation of MIC precursor form and a decrease in the secreted mature form, resulting in serious defects in invasion and egress [29]. Moreover, for Plasmodium and Cryptosporidium, SUB1 is the essential molecule that directly affects the egress process [30, 31]. Although, the function of NcSUB1 has never been identified, NcSUB1 has an Apicomplexa conserved subtilisin SUB1-like catalytic domain, and its homology with TgSUB1 is 79.3%.

The secretion assay results further confirmed that the decrease in the transcription level of SUB1 in Δncgra41 strain led to a significant reduction in the secretion of NcMIC1 into the supernatant, impacting the egress and motility of N. caninum. Moreover, whether NcGRA41 directly interact with NcSUB1 requires further investigation. The secretion levels of NcMIC4 and NcMIC8 did not change significantly; it is possible that the stimulation time for the secretion test was too long, making the observed difference not statistically significant. In addition, other factors that affect the egress process may exist but were not detected through microneme secretionassays.

In addition to TgGRA41, TgGRA22 has also been reported to be associated with the egress of T. gondii. Previous research has identified the Neospora homologous gene NcGRA22, although its biological function has not been studied yet. If it can be confirmed that NcGRA22 also affects the egress process, future work will focus on elucidating the specific contributions of NcGRA22 and NcGRA41 on the egress process by constructing double knockout strains.

In summary, our study identified a novel dense granule protein from N. caninum, named NcGRA41. NcGRA41 was found to play a crucial role in the regulation of tachyzoites egress, impacting the pathogenicity of the parasites. This discovery contributes to a better understanding of the functions of Neospora GRAs, and NcGRA41 emerges as a potential virulence factor or vaccine candidate gene for N. caninum.

Acknowledgments

We thank Professor Xuenan Xuan (Obihiro University of Agriculture and Veterinary Medicine, Japan) for kindly providing the Nc1 strain. We are grateful to Professor Silvia N. J. Moreno (University of Georgia, USA) for providing the pTCR vector. We are grateful to Professor Bang Shen (Huazhong Agricultural University) for kindly providing CRISPR/CAS9-UPRT vector.

Funding

This work was supported by funds of National Natural Science Foundation of China (32273029 and 31972700).

Availability of data and materials

The data generated or analyzed during this wok are included within this paper.

Authors’ contributions

J.Y, YQ.P, Q.L and J.L conceived and designed this research together; J.Y and YQ.P cooperated to conduct the experiment and analyze experimental data; XM.W, Z.Y, ZF.Z helped to draft the manuscript, J.L revised the manuscript critically. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The mice used in this study were handled in compliance with the recommendations outline in the "Guidelines for the Care and Use of Laboratory Animals" of the Ministry of Science and Technology of China. Additionally, our research protocol received approval from the Beijing Laboratory Administration Committee.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflicts of interest.

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No competing interests reported.

TableS1.xlsx
Additional file 1: Table S1. Primers used in this study
Fig.S1.tif
Additional file 2: Fig.S1 Construction and identification of Δncgra41 and comΔncgra41. a Schematic diagram illustrating the construction of pTCR-NcGRA41 and pCRISPR/Cas9-ΔNcGRA41. b PCR identification of Δncgra41 monoclonal strains.1-11: Monoclonal strain, 12: Nc1, 13: Water, 14: Mixed strain, M: 2000 AL. c Schematic diagram illustrating the construction of pCRISPR/Cas9-comΔncgra41. d PCR identification of comgra41 monoclonal strains. 1: Mixed strain, 2-23: Monoclonal strain, M: 2000 AL. e Subcellular localization of comΔncgra41 by IFA. Red outlines tachyzoites labeled with rabbit anti-NcSRS2 polyclonal antibody, Green represents comΔncgra41 strain labeled with mouse anti-HA monoclonal antibody, Nuclei were labeled with Hoechst. Scale bar = 5 μm.

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Role of GRA41 in Neospora caninum pathogenicity: insights into tachyzoite egress and microneme secretion

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Parasites and cell cultures

Generation of transgenic strains

Western blot

Immunofluorescence assay

Plaque assay

Invasion assay

Proliferation assay

Egress assay

Gliding assay

Parasite virulence assay

Quantitative real-time PCR

Microneme secretion assay

Statistical analysis

Results

NcGRA41-deficient parasites delay the egress of tachyzoites and reduce virulence

Deletion of NcGRA41 affects secretion and expression of some NcMICs

NcGRA41 deletion affects the transcription level of SUB1 genes

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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