Edição genômica por CRISPR/Cas9 aplicada à identificação e caracterização funcional da proteína de grânulo denso 5 de Neospora caninum

Abstract

Identified in 1988, Neospora caninum is an obligate intracellular parasite belonging to the phylum Apicomplexa. It is the etiological agent of neosporosis, a disease responsible for causing abortions in cattle and neuromuscular disorders in dogs. Its life cycle is heteroxenous, involving horizontal transmission through the ingestion of non-sporulated oocysts shed in the feces of canids, as well as infected tissue cysts, and vertical transmission via the transplacental passage of tachyzoites. Among the dozens of proteins involved in the invasion process, dense granule proteins (GRAs) stand out due to their roles in the parasite’s lytic cycle, nutrient acquisition, and/or modulation of the host immune response. Although they are of recognized relevance, the GRAs of N. caninum still lack in-depth characterization, highlighting the need for further investigations from this perspective. The aim of this study was to identify and characterize the dense granule protein 5 (GRA5) of N. caninum. To identify and localize this protein for the first time, we performed a genetic modification using CRISPR/Cas9 targeting the NcGRA5 coding region, which induced the expression of three viral hemagglutinin epitopes at the C-terminal portion of the protein. Using this genetically modified parasite line, we demonstrated that NcGRA5 displays a distribution pattern within the parasitophorous vacuole that is typical of dense granule proteins. In addition, our data estimate that NcGRA5 dimers have approximate molecular weights of 32 kDa and 26 kDa. In order to investigate the biological function of this protein, we generated parasites genetically deficient in NcGRA5. The replication capacity of Δgra5 parasites was evaluated in comparison with the parental strain using plaque assays in HFF fibroblasts. Deletion of the NcGRA5 gene significantly reduced parasite replication in vitro, as evidenced by a decrease in the number of lytic plaques formed on fibroblast monolayers compared with parental parasites. In this context, our findings expand current knowledge on dense granule proteins of N. caninum, demonstrating that this protein is involved in the parasite’s replicative capacity and highlighting NcGRA5 as a potential target for future studies aimed at infection control.