Rhoptry protein 5 (ROP5) Is a Key Virulence Factor in Neospora caninum

Ma, Lei; Liu, Jing; Li, Muzi; Fu, Yong; Zhang, Xiao; Li, Qun

doi:10.3389/fmicb.2017.00370

Cited by 23 publications

(21 citation statements)

References 60 publications

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“…So we must understand these phenotypes in future study. A recent study showed that the loss of NcROP5 by N. caninum led to a reduction in mouse mortality and reduced NcGRA7 transcription in the parasite (12). According to ToxoDB, sequences that share high identity with TgROP5 (TgROP5A, TgROP5B, and TgROP5C) have been detected the genome of the N. caninum Liverpool strain: NcLIV_060730, NcLIV_060740, and NcLIV_060741, respectively (12,29).…”

Section: Discussionmentioning

confidence: 99%

“…A recent study showed that the loss of NcROP5 by N. caninum led to a reduction in mouse mortality and reduced NcGRA7 transcription in the parasite (12). According to ToxoDB, sequences that share high identity with TgROP5 (TgROP5A, TgROP5B, and TgROP5C) have been detected the genome of the N. caninum Liverpool strain: NcLIV_060730, NcLIV_060740, and NcLIV_060741, respectively (12,29). On the contrary, RNA sequencing in this study showed no significant differences in the expression levels of Neospora ROP5 in Nc1 and the NcGRA7-deficient and NcGRA7-complemented lines (see Table S1 in the supplemental material).…”

Section: Discussionmentioning

confidence: 99%

“…Several proteins of N. caninum have been identified as effector molecules that could interact with host signaling pathways. The rhoptry proteins of N. caninum, NcROP5 and NcROP16, may be virulence factors because parasites deficient in these proteins cause reduced mortality in mice (12,13). NcROP16 is also responsible for STAT3 activation (13).…”

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confidence: 99%

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Neospora caninum Dense Granule Protein 7 Regulates the Pathogenesis of Neosporosis by Modulating Host Immune Response

Nishikawa

Shimoda

Fereig

et al. 2018

Appl Environ Microbiol

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is a protozoan parasite closely related to Neosporosis caused by is considered one of the main causes of abortion in cattle and nervous-system dysfunction in dogs, and identification of the virulence factors of this parasite is important for the development of control measures. Here, we used a luciferase reporter assay to screen the dense granule proteins genes of , and we found that NcGRA6, NcGRA7, and NcGRA14 are involved in the activation of the NF-κB, calcium/calcineurin, and cAMP/PKA signals. To analyze the functions of these proteins and cyclophilin, we successfully knocked out their genes in the Nc1 strain using plasmids containing the CRISPR/Cas9 components. Among the deficient lines, the -deficient parasites showed reduced virulence in mice. An RNA sequencing analysis of infected macrophage cultures showed that NcGRA7 mainly regulates the host cytokine and chemokine production. The levels of gamma interferon in the ascites fluid, CXCL10 expression in the peritoneal cells, and CCL2 expression in the spleen were lower 5 days after infection with the-deficient parasite than after infection with the parental strain. The parasite burden and the degree of necrosis in the brains of mice infected with the -deficient parasite were also lower than in those of the parental strain. Collectively, our data suggest that both the NcGRA7-dependent activation of the inflammatory response and the parasite burden are important in virulence. invades and replicates in a broad range of host species and cells within those hosts. The effector proteins exported by induce its pathogenesis by modulating the host immunity. We show that most of the transcriptomic effects in-infected cells depend upon the activity of NcGRA7. A deficiency in NcGRA7 reduced the virulence of the parasite in mice. This study demonstrates the importance of NcGRA7 in the pathogenesis of neosporosis.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Neospora caninum Dense Granule Protein 7 Regulates the Pathogenesis of Neosporosis by Modulating Host Immune Response

Nishikawa

Shimoda

Fereig

et al. 2018

Appl Environ Microbiol

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“…After identification at the genomic level, protein level, transcription level, and protein distribution, we verified that the NcGRA6 strain was successfully generated and could be used for further NcGRA6 gene function research. In N. caninum, the NcSRS2 protein was used as a surface protein in GRA17 and ROP5 gene knockout strains (31,34). During identification of the NcGRA6 strain at the protein level, we first labeled the specific putative surface protein NcSRS22A in N. caninum using its monoclonal antibody (identified and prepared in our laboratory) to explore the location of GRA6 protein.…”

Section: Discussionmentioning

confidence: 99%

“…To further explore the influence of NcGRA6 in N. caninum virulence, we conducted experiments both in vitro and in vivo. Plaque, invasion, egress, and replication assays are common technologies for the applied in in vitro parasite virulence research (31,34). In our study, we performed these assays to compare virulence changes between the N. caninum NcGRA6 strain and Nc-1 strain.…”

Section: Discussionmentioning

confidence: 99%

Effects of Dense Granular Protein 6 (GRA6) Disruption on Neospora caninum Virulence

et al. 2020

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Neospora caninum (N. caninum) is a major cause of abortions in cattle. During its invasion of host cells, a parasitophorous vacuole (PV) is formed, accompanied by an intravacuolar network (IVN). The IVN takes part in parasite ingesting of nutrients from hosts. The dense granular proteins of N. caninum (NcGRAs) play a key role in forming the PV and the IVN, which may influence virulence during N. caninum invasion. The present study aimed to explore the biological function of NcGRA6 in N. caninum by disrupting the NcGRA6 gene in the Nc-1 strain. We successfully constructed an NcGRA6-targeting CRISPR plasmid (pNc-SAG1-Cas9:U6-SgGRA6) and amplified the DHFR-TS DNA donor. The NcGRA6 knockout mutation (NcGRA6) was generated by co-electroporation of the pNc-SAG1::CAS9-U6::sgGRA6 plasmid and the DHFR-TS DNA donor into the Nc-1 strain, which was then cultured under pyrimethamine selection pressure. The NcGRA6 mutation was further verified by identification of NcGRA6 gene disruption using PCR, measurement of NcGRA6 gene transcription levels using qPCR, assessment of NcGRA6 protein expression levels using western blotting, and observation of NcGRA6 protein location using immunofluorescence and immunoelectron microscopy. The results of in vitro virulence assays, including plaque, invasion, egress, and replication assays, showed that the NcGRA6 strain had smaller plaques, similar invasion and egress ability, and slower intracellular replication ability than the Nc-1 strain. The results of in vivo virulence assays showed that the NcGRA6 strain exhibited reduced virulence and improved survival ability in mice compared with the Nc-1 strain. The parasite burden in NcGRA6 strain-infected mouse tissues, including the heart, brain, liver, spleen, lung, and kidney, was significantly reduced compared with that in mice infected with the Nc-1 strain. These data suggest that we successfully constructed a NcGRA6 strain and verify that NcGRA6 is a critical virulence factor. NcGRA6 gene disruption can slow down N. caninum proliferation and lower the pathogenicity to hosts. Our findings provide a foundation for future research on other targeted N. caninum protein functions and may help in exploring the interaction mechanisms between parasites and hosts.

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