Caracterização de uma nova proteína de grânulo denso de Neospora caninum utilizando a técnica de CRISPR/Cas9

Abstract

Neospora caninum is a parasite from the phylum Apicomplexa, known for causing neosporosis, primarily affecting pregnancies in cattle and leading to neuromuscular dysfunction in dogs. The parasite's life cycle involves both horizontal transmission, through the ingestion of cysts, and vertical transmission, via transplacental transfer of tachyzoites. Dense granule proteins (GRAs) play a crucial role in cell invasion processes and immune response modulation, being recognized as essential components in the biology of Apicomplexa parasites. Despite their importance, the GRAs of N. caninum have not yet been sufficiently characterized, highlighting the need for further studies in this area. The objective of this project was to characterize the NCLIV_060220 protein, predicted to be a GRA, through bioinformatics techniques and CRISPR/Cas9 genome editing, generating genetically modified parasites in order to elucidate the role of this protein in the parasite-host interaction. To this end, cell culture assays were used, along with immunofluorescence to determine the localization of the target protein, Western blotting to analyze the protein profile, and quantitative PCR to measure RNA levels in a tachyzoite kinetics assay. From the results obtained, the presence of a conserved bacterial domain shared among orthologs in algae and apicomplexans was identified. Additionally, the absence of transmembrane domains was observed. However, a signal peptide and cleavage site were detected. Finally, increased mRNA expression was noted during the initial invasion period, followed by a subsequent decline. Immunofluorescence revealed localization of the protein within the vacuole in the extracellular space, and greater protein expression was observed after 18 hours of infection. Furthermore, Western blotting results showed the presence of two bands, indicating the beginning of post-translational processes. These analyses are paving the way for a future functional study through gene deletion.