Person:
García Sánchez, Marta Eulalia

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First Name
Marta Eulalia
Last Name
García Sánchez
URI
https://hdl.handle.net/20.500.14352/80623
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Veterinaria
Department
Sanidad Animal
Area
Sanidad Animal
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2022 - 20232
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Author: Amieva, Rafael ×

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    Characterization of Neospora caninum virulence factors NcGRA7 and NcROP40 in bovine target cells
    (Veterinary Parasitology, 2023) Rico San Román, Laura; Amieva, Rafael; Horcajo Iglesias, María Del Pilar; García Sánchez, Marta Eulalia; Pastor Fernández, Iván; Ortega Mora, Luis Miguel; Collantes Fernández, Esther
    Bovine neosporosis is one of the major causes of reproductive failure in cattle worldwide, and differences in virulence between isolates have been widely shown. However, the molecular basis and mechanisms underlying virulence in Neospora caninum are mostly unknown. Recently, we demonstrated the involvement of NcGRA7 and NcROP40 in the virulence of N. caninum in a pregnant murine model using single knockout mutants in these genes generated by CRISR/Cas9 technology. In this study, the role of these proteins was investigated in two in vitro models using bovine target cells: trophoblast (F3 cell line) and monocyte-derived macrophages (BoMØ). The proliferation capacity of the single knockout mutant parasites was compared to the wild-type strain, the Nc-Spain7 isolate, using both cell populations. For the bovine trophoblast, no differences were observed in the growth of the defective parasites compared to the wild-type strain, neither in the proliferation kinetics nor in the competition assay. However, in naïve BoMØ, a significant decrease in the proliferation capacity of the mutant parasites was observed from 48 h pi onwards. Stimulation of BoMØ with IFN-γ showed a similar inhibition of tachyzoite growth in defective and wild-type strains in a dose-dependent manner. Finally, BoMØ infected with knockout parasites showed higher expression levels of TLR3, which is involved in pathogen recognition. These results suggest that NcGRA7 and NcROP40 may be involved in the manipulation of innate immune defense mechanisms against neosporosis and confirm the usefulness of the BoMØ model for the evaluation of N. caninum virulence mechanisms. However, the specific functions of these proteins remain unknown, opening the way for future research.
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    NcGRA7 and NcROP40 Play a Role in the Virulence of Neospora caninum in a Pregnant Mouse Model
    (Pathogens, 2022) Rico San Román, Laura; Amieva, Rafael; Regidor Cerrillo, Javier; García Sánchez, Marta Eulalia; Collantes Fernández, Esther; Pastor Fernández, Iván; Saeij, Jeroen P. J.; Ortega Mora, Luis Miguel; Horcajo Iglesias, María Del Pilar
    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.