Molecular characterization of NCLIV_011700 of Neospora caninum, a low sequence identity rhoptry protein

Abstract

Neospora caninum is an obligate intracellular parasite related to abortion in cattle, goats and sheep. The life cycle of N. caninum is characterized by the time-coordinated secretion of proteins contained in micronemes, rhoptries and dense granules, allowing the active invasion and the adaptation of the parasite in the cell environment. Thus, the proteins of the secretome have the potential to be considered as targets for N. caninum control. Despite the importance of neosporosis in the livestock-related economy, no commercial treatment is available. Furthermore, the process of invasion, propagation and immune evasion are not completely elucidated. In this study, we initiated the characterization of NCLIV_011700 of N. caninum, a protein with low sequence identity to NcROP15 or TgROP15 (<15%). Our goal was the detection and molecular characterization of the NCLIV_011700, once homology (with low identity >20%) was observed within the Apicomplexa. The NCLIV_011700 sequence was aligned and compared to the closer apicomplexan homologues (ROP15 from N. caninum, T. gondii, Hammondia hammondi, Cystospores suis), including the predicted domains. In general, the NCLIV_011700 demonstrated low identity with ROP15 of apicomplexan (<20%) and had a ubiquitin domain. On the other side, the NCLIV_011700 homologues were composed of a non-cytoplasmic domain, suggesting different functions between NcROP15 (or homologues) and NCLIV_011700 during the parasite life cycle. Moreover, the NCLIV_011700 was amplified by PCR, ligated to a pET28a plasmid and expressed in Escherichia coli. The recombinant form of NCLIV_011700 was purified in a nickel-Sepharose resin and applied for polyclonal antibody production in mice. The antiserum against NCLIV_011700 (anti-rNCLIV_011700) was used to localize the native form of the protein using Western blot and confocal microscopy. Also, the NCLIV_011700 antiserum partially inhibited the parasite adhesion/invasion process, indicating an active role of the protein in the N. caninum cycle. Thus, the initial NCLIV_011700 characterization will contribute to enlarging the comprehension of N. caninum, aiming at the future development of tools to control the parasite infection/propagation.