Disruption of Dense Granular Protein 2 (GRA2) Decreases the Virulence of Neospora caninum

Dong, Jingquan; Zhang, Nan; Zhao, Panpan; Li, Jianhua; Cao, Lili; Wang, Xiaocen; Li, Xin; Ju, Yang; Zhang, Xichen

doi:10.3389/fvets.2021.634612

Cited by 8 publications

(4 citation statements)

References 59 publications

(77 reference statements)

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“…In T. gondii, many of these effector proteins have been described as virulence factors [24,[50][51][52]. Although the understanding of the pathogenic molecular mechanisms in N. caninum is not as extensive as in the closely related T. gondii, some rhoptry (ROP16, ROP5) and dense granule (GRA7, GRA17, GRA6, GRA2) proteins have already been described as virulence factors in N. caninum [31,35,36,[53][54][55].…”

Section: Discussionmentioning

confidence: 99%

“…In this study, the Nc∆ROP40 strain was generated following the same approach that was used for the generation of the NcGRA7-deficient strain [39]. In contrast to other studies that failed or had very low efficiency disrupting genes in N. caninum using the tools developed for T. gondii [53][54][55], we successfully generated the KO and complemented lines using this system. In addition, we used the highly virulent and well-characterised isolate Nc-Spain 7 instead of Nc-1.…”

Section: Discussionmentioning

confidence: 99%

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NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model

et al. 2022

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model

et al. 2022

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“…To obtain VSPAS7 protein, the pET-28a- vspas7 expression vector was induced and purified. Two-month-old New Zealand female rabbits were immunized two times at 14-day intervals by using an equal mixture of VSPAS7 protein and Freund's adjuvant [ 31 ]. After vaccination, rabbit serum was collected, and anti-VSPAS7 antibody was obtained by measuring antibody efficacy.…”

Section: Methodsmentioning

confidence: 99%

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis

Sun,

Zhao,

et al. 2023

Parasites Vectors

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Background The unicellular protozoan parasite Giardia intestinalis, which primarily infects humans and animals such as cattle and sheep, is having a major negative impact on public health. Giardia is able to evade the recognition and elimination of the host immune system because of the trophozoite surface and extracellular vesicles (EVs) covered by variant-specific surface proteins (VSPs). As key proteins for immune evasion, whether VSPs can regulate Giardia-induced pyroptosis and promote Giardia evasion of host immune responses has not been reported. Methods To examine the role of Giardia VSPAS7 on Giardia-induced activation of the signaling pathway, secretion of pro-inflammatory cytokines, pyroptosis and the mechanism involved, we constructed the pcDNA3.1-vspas7 expression plasmid and transfected this plasmid into mouse macrophages. Key proteins for pyroptosis, IL-1β secretion and LDH release were detected in pcDNA3.1-vspas7-transfected wild-type (WT) cells and NLRP3-deficient cells by western blot, ELISA and LDH assays, respectively. The interactions of Giardia VSPAS7 and mouse NLRP3 were examined using immunofluorescence assays (IFA), co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. Results VSPAS7 could decrease the levels of phosphorylated-p65 (P-p65), P-IκBα and P-ERK caused by Giardia and reduce the production levels of Giardia-induced pro-inflammatory cytokine IL-6, IL-12 p40 and TNF-α. The results showed that VSPAS7 inhibited Giardia-mediated activation of NF-κB, ERK/MAPK signaling and secretion of pro-inflammatory cytokines. Furthermore, VSPAS7 suppressed Giardia-induced macrophage pyroptosis by reducing GSDMD cleavage, caspase-1 activation, IL-1β secretion and LDH release. We further found that VSPAS7 could interact with mouse NLRP3 directly, and in NLRP3-deficient cells the suppression of Giardia-induced macrophage pyroptosis by VSPAS7 was significantly attenuated. Conclusions Overall, VSPAS7 could inhibit Giardia-induced activation of signaling pathways and pyroptosis in host macrophages, allowing Giardia evasion of host immune responses. Studies on Giardia VSP-mediated immune evasion provide an important theoretical basis for in-depth studies on Giardia pathogenicity. Graphical abstract

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“…Subsequently, an increased number of mutant parasites deficient in certain genes have been developed by disrupting the target gene sequence by incorporation of a selection marker. The most frequently used selection marker is the modified dihydrofolate reductase-thymidylate synthase (mdhfr-ts) (Nishikawa et al, 2018;Zhao et al, 2020;Dong et al, 2021;Wang et al, 2021;Yang et al, 2021), which provides resistance to pyrimethamine, allowing the selection of mutant parasites that grow in the presence of pyrimethamine.…”

Section: Introductionmentioning

confidence: 99%

TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers

Rico-San Román,

Hänggeli,

Hemphill

et al. 2024

Front. Cell. Infect. Microbiol.

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As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

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Disruption of Dense Granular Protein 2 (GRA2) Decreases the Virulence of Neospora caninum

Cited by 8 publications

References 59 publications

NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model

NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model

Giardia VSPAS7 protein attenuates Giardia intestinalis-induced host macrophage pyroptosis

TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers

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