Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures

Vonlaufen, Nathalie; Müller, Norbert; Keller, N.; Naguleswaran, Arunasalam; Bohne, Wolfgang; McAllister, Milton M.; Björkman, Camilla; Müller, Eliane J.; Caldelari, Reto; Hemphill, Andrew

doi:10.1016/s0020-7519(02)00126-1

Cited by 62 publications

(50 citation statements)

References 44 publications

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“…BAG1, a classical bradyzoite-specific marker (McAllister et al 1996;Vonlaufen et al 2002Vonlaufen et al , 2004, was used to monitor the conversion efficiency. For this purpose, parasites were purified from SNP-treated cells and the expression levels of BAG1 and BAG1 mRNA were compared with those of tachyzoites by immunofluorescence (IF) and quantitative RT-PCR (qRT-PCR), respectively ( Fig.…”

Section: Cell and Parasite Culturesmentioning

confidence: 99%

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

et al. 2013

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S U M M A R YRecent publications demonstrated that a fragment of a Neospora caninum ROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein, N. caninum ROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of 'cysts' produced in vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasion in vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

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Section: Cell and Parasite Culturesmentioning

confidence: 99%

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

et al. 2013

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“…The major T. gondii surface antigens TgSAG1 and TgSAG2 are stage-specifically expressed in tachyzoites (30). Similarly, the two major immunodominant N. caninum tachyzoite surface antigens, NcSAG1 and NcSRS2 (9,10,48), were observed to be down-regulated during tachyzoite-to-bradyzoite stage conversion (17,47,55). Several bradyzoite-specific T. gondii antigens, such as TgBAG1 (5,42), have been identified (6,49,52), and polyclonal antibodies directed against recombinant TgBAG1 were shown to cross-react with bradyzoites of N. caninum (36,53,55,58).…”

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confidence: 97%

“…Several bradyzoite-specific T. gondii antigens, such as TgBAG1 (5,42), have been identified (6,49,52), and polyclonal antibodies directed against recombinant TgBAG1 were shown to cross-react with bradyzoites of N. caninum (36,53,55,58). Furthermore, the monoclonal antibody MAbCC2, reacting with a 115-kDa T. gondii cyst wall protein (18), was recently demonstrated to cross-react with N. caninum tissue cysts (31,55). In addition to stage-specifically expressed antigens, T. gondii dense granule proteins, which are secreted shortly after invasion and which are involved in the modification of the parasitophorous vacuole (11), have been shown to be differentially located in T. gondii tachyzoite and bradyzoite cysts (52).…”

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In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites

et al. 2004

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We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 M sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.

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“…At the same time, Th1 cytokines such as IFN-γ result in the activation of pathways that generate NO (Liu et al, 2004), which is potentially lethal to many protozoa (Hunter and Reiner, 2000). Mice depleted of the gene encoding inducible NO synthase, which mediates the formation of NO, were more susceptible to N. caninum infection than wild-type mice (Tanaka et al, 2000;Vonlaufen et al, 2002). In the present study, the groups immunised with the DNA vaccine with or without adjuvant had significantly higher plasma levels of NO than the saline or pcDNA3.1/Zeo (+) vector controls, consistent with the increase in the expression of cytokines.…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity of a DNA vaccine expressing the Neospora caninum surface protein NcSRS2 in mice

Zhao

Ding

et al. 2009

Acta Veterinaria Hungarica

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The immunogenicity of a DNA vaccine expressing the surface protein NcSRS2 of Neospora caninum was studied in BALB/c mice. The NcSRS2-encoding DNA was obtained by PCR amplification of the NcSRS2 ORF gene from the p43 plasmid encoding the N. caninum surface protein NcSRS2, ligated to the mammalian expression vector pcDNA3.1/Zeo(+) and propagated in E. coli DH5α to produce the N. caninum NcSRS2 DNA vaccine. BALB/c mice were immunised by two intramuscular injections of the DNA vaccine with or without complete Freund's adjuvant (CFA). Serum antibody titres and nitric oxide (NO) concentrations, and splenocyte proliferation and cytokine expression were measured after immunisation. The DNA vaccine induced T-cell-mediated immunity as shown by significantly increased NO concentrations, cytokine gene (IL-2 and IFN-γ) expression, and NcSRS2 protein-stimulated lymphocyte proliferation in mice immunised with the DNA vaccine. The vaccine also induced weak humoral immunity. The immunogenicity of the DNA vaccine was slightly enhanced by CFA. The immune response was specific to NcSRS2. No immune response was observed in mice immunised with the pcDNA3.1/Zeo(+) vector alone.

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Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures

Cited by 62 publications

References 44 publications

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites

Immunogenicity of a DNA vaccine expressing the Neospora caninum surface protein NcSRS2 in mice

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