Nc GRA7 and Nc ROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model

Abstract

The intraspecific variability among Neospora caninum isolates in their in vitro behaviour and in vivo virulence has been widely studied. In particular, transcriptomic and proteomic analyses have shown a higher expression/abundance of specific genes/proteins in high-virulence isolates. Consequently, the dense granule protein NcGRA7 and the rhoptry protein NcROP40 were proposed as potential virulence factors. The objective of this study was to characterize the role of these proteins using CRISPR/Cas9 knockout (KO) parasites in a well-established pregnant BALB/c mouse model of N. caninum infection at midgestation. The deletion of NcGRA7 and NcROP40 was associated with a reduction of virulence, as infected dams displayed milder clinical signs, lower parasite burdens in the brain, and reduced mortality rates compared to those infected with the wild-type parasite (Nc-Spain7). Specifically, those infected with the NcGRA7 KO parasites displayed significantly milder clinical signs and a lower brain parasite burden. The median survival time of the pups from dams infected with the two KO parasites was significantly increased, but differences in neonatal mortality rates were not detected. Overall, the present study indicates that the disruption of NcGRA7 considerably impairs virulence in mice, while the impact of NcROP40 deletion was more modest. Further research is needed to understand the role of these virulence factors during N. caninum infection.

Keywords: BALB/c; CRISPR/Cas9; NcGRA7 protein; NcROP40 protein; Neospora caninum; pregnant mouse model; virulence factor.

Figure 1

Construction and verification of the knockout and complemented strains by CRISPR/Cas9. (A) Schematic diagram of NcROP40 gene disruption and the repair template in Nc-Spain7 (wild-type). Disruption was achieved by employing two guide RNAs (g1 and g2). The positions of primers used for diagnostic PCR (P1–6) are indicated by arrows. (B) Schematic diagram of NcROP40 and NcGRA7 complementation of the knockout parasites by insertion at the UPRT locus. The UPRT gene was disrupted employing one guide RNA (g1). The positions of primers used for diagnostic PCR (P5–8) are indicated by arrows. UTR, untranslated region; DHFR–TS, dihydrofolate reductase–thymidylate synthase; LoxP: loxP sites.

Figure 2

Verification of the knockout and complemented strains. (A) Diagnostic PCR demonstrating the loss of NcROP40 (P1–P2, P5–P6) and the integration and orientation of the donor template (P2–P4, P1–P3) into the correct locus in the NcROP40 knockout parasite. Diagnostic PCR demonstrating the integration of NcROP40 (P5–P6) and NcGRA7 (P7–P8) in the complemented strains. Primers used for PCR are described in Table 1 and indicated in Figure 1. (B) Western blotting assessment of NcROP40 and NcGRA7 gene disruption and complementation by measuring the protein expression levels. Anti-NcROP40 rabbit serum detected a 53-kDa protein in the WT and complemented strain (iNc∆ROP40) but not in the knockout (Nc∆ROP40) strain. A rabbit anti-NcGRA7 antibody detected two major proteins of 33 and 18 kDa in the WT and in the complemented strain (iNc∆GRA7), but not in the knockout parasite (Nc∆GRA7). (C) Immunofluorescence staining of Marc-145 cells infected with different strains of Neospora caninum (WT, knockouts, and complemented strains). The nuclei were stained with DAPI (blue), and the monoclonal antibody α-NcSAG1 was used as a control to mark the periphery of the parasites (red). The images above show methanol-fixed cultures labelled with affinity purified polyclonal antibody α-NcROP40 (green) and the images below show paraformaldehyde/glutaraldehyde-fixed cultures labelled with affinity purified polyclonal antibody α-NcGRA7 (green). White arrows indicate the expression or lack of expression of the proteins NcROP40 or NcGRA7 into the parasitophorous vacuole. Note that NcROP40 and NcGRA7 protein are detected in the WT and in the complemented strains but not in the knockout strains. WT, wild type (NcSpain7); NC, negative control; NcΔROP40, NcROP40 knockout parasite; NcΔGRA7, NcGRA7 knockout parasite; iNcΔROP40, complemented strain of NcROP40 knockout parasite; iNcΔGRA7, complemented strain of NcGRA7 knockout parasite.

Figure 3

Effect of Neospora caninum infection in the offspring during a period of 30 days pp (postpartum). Kaplan–Meier survival curves of pups from dams infected on Days 7 of pregnancy with 10⁵ tachyzoites from different N. caninum strains and the uninfected group (negative control, inoculated with PBS). The wild-type (WT) group was challenged with the Nc-Spain7 isolate, and the NcΔGRA7 and NcΔROP40 groups were infected with knockout mutant parasites (deficient in GRA7 and ROP40 proteins, respectively). Each point represents the percentage of surviving animals on that day, and vertical steps downwards correspond to observed deaths. Letters a, b, c and d indicate significant differences (p < 0.05, log-rank test). Note that a delay in time to death was observed in groups infected with mutant parasites compared to the WT group.

Figure 4

Clinical signs of dams infected with Neospora caninum tachyzoites (10⁵ tachyzoites/mouse). The wild-type (WT) group was infected with the Nc-Spain7 isolate, and the NcΔGRA7 and NcΔROP40 groups were infected with knockout mutant parasites (deficient in GRA7 and ROP40 proteins, respectively). The negative control was challenged with PBS. Scores were based on the detection and severity of clinical signs (0, no alterations; 1, ruffed coat; 2, rounded back; 3, severe weight loss or 4, nervous signs). Each point represents a single animal. Significant differences between infected groups are denoted by horizontal lines and asterisks (* p < 0.05; Kruskal–Wallis Dunn’s comparison post-test).

Figure 5

Cerebral parasite burden at 30 Days postinfection in mice infected with 10⁵ tachyzoites of different Neospora caninum strains and the uninfected group (negative control). The wild-type (WT) group was challenged with the Nc-Spain7 isolate, and the NcΔGRA7 and NcΔROP40 groups were infected with knockout mutant parasites (deficient in GRA7 and ROP40 proteins, respectively). Each dot represents individual values, and medians are represented as horizontal lines. Parasite burden in the brain was calculated using real-time qPCR. (A) Parasite burden in the brains of dams infected on Day 7 of gestation. (B) Parasite burden in the brains of nonpregnant mice. Significant differences between infected groups are denoted by horizontal lines and asterisks (* p < 0.05, Kruskal–Wallis Dunn’s comparison post-test).

Figure 6

Cellular immune response in Neospora caninum-infected mice during the acute phase (5 days postinfection). The wild-type (WT) group was challenged with the Nc-Spain7 isolate, and the NcΔGRA7 and NcΔROP40 groups were infected with knockout mutant parasites (deficient in GRA7 and ROP40 proteins, respectively). Box-plot graphs represent the cytokine expression in the spleen, the mean (horizontal lines), the lower and upper quartiles (boxes) and minimum and maximum values (whiskers). Each sample was normalized to β-actin expression and relative to the negative control group. The results are given by 2^−∆∆Ct, and the comparative threshold cycle method was used. According to the Kruskal–Wallis test, no significant differences were present between infected groups.

Figure 7

Humoral immune responses in challenged mice at 30 days postinfection. Anti-N. caninum immunoglobulins (IgG1 and IgG2a isotypes) generated in (A) dams and (B) nonpregnant mice inoculated with 10⁵ tachyzoites from different N. caninum strains and the uninfected group (PBS). The wild-type (WT) group was challenged with the Nc-Spain7 isolate, and the NcΔGRA7 and NcΔROP40 groups were infected with knockout mutant parasites (deficient in GRA7 and ROP40 proteins, respectively). Bars represent the average RIPC (relative index percent), and error bars represent standard deviations for each group. Significant differences between infected groups are denoted by horizontal lines and asterisks with horizontal lines (*** p < 0.001; ** p < 0.01; * p < 0.05, one-way ANOVA Tukey’s comparison post-test).