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Cloning and expression of Neospora caninum dense-granule 7 in E. coli.

ABSTRACT: Neospora caninum is an obligate intracellular protozoan which induces abortion, still birth and neuromuscular disorders in cattle and is an important problem in dairy and beef industry worldwide. The dense granule protein 7 (GRA7) of N. caninum is an immune-dominant protein shared by both tachyzoite and bradyzoites. This study was conducted to produce recombinant GRA7 of N. caninum using a plasmid with high level of expression of this protein in E. coli. For this purpose, a segment of N. caninum DNA corresponding to GRA7 was amplified using PCR. After sequencing, this fragment was cloned into expression vector pMAL-c2X under the control of the lac promoter. Expression of this plasmid in E. coli strain TG1 was identified by western blotting. In this study, pMAL-c2X had a strong promoter to produce high level expression of NcGRA7. This result revealed that this recombinant protein with pMAL-c2X vector may be suitable for developing of diagnostic procedures.

SUBMITTER: Kefayat M

PROVIDER: S-EPMC4000374 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Cloning and expression of Neospora caninum dense-granule 7 in E. coli.

Kefayat Marziye M Hamidinejat Hossein H Seifiabadshapoori Masoud Reza MR Namavari Mohammad Mehdi MM Shayan Parviz P Gooraninejad Saad S

Journal of parasitic diseases : official organ of the Indian Society for Parasitology 20121218 2

Neospora caninum is an obligate intracellular protozoan which induces abortion, still birth and neuromuscular disorders in cattle and is an important problem in dairy and beef industry worldwide. The dense granule protein 7 (GRA7) of N. caninum is an immune-dominant protein shared by both tachyzoite and bradyzoites. This study was conducted to produce recombinant GRA7 of N. caninum using a plasmid with high level of expression of this protein in E. coli. For this purpose, a segment of N. caninum ...[more]

PMID: 24808652

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Project description:Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii Neosporosis caused by N. caninum 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 N. caninum, 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 Neospora cyclophilin, we successfully knocked out their genes in the Nc1 strain using plasmids containing the CRISPR/Cas9 components. Among the deficient lines, the NcGRA7-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 NcGRA7-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 NcGRA7-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 Neospora virulence.IMPORTANCENeospora caninum invades and replicates in a broad range of host species and cells within those hosts. The effector proteins exported by Neospora induce its pathogenesis by modulating the host immunity. We show that most of the transcriptomic effects in N. caninum-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.

Project description:BackgroundNTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided.MethodsHerein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points.ResultsWe describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress.ConclusionsThe present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle.

Project description: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 (ΔNcGRA6) 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 ΔNcGRA6 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 ΔNcGRA6 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 ΔNcGRA6 strain exhibited reduced virulence and improved survival ability in mice compared with the Nc-1 strain. The parasite burden in ΔNcGRA6 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 ΔNcGRA6 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.

Dataset Information

Cloning and expression of Neospora caninum dense-granule 7 in E. coli.

Publications

Cloning and expression of Neospora caninum dense-granule 7 in E. coli.

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