Characterization of aspartyl aminopeptidase from Toxoplasma gondii

Aminopeptidases have emerged as new promising drug targets for the development of novel anti-parasitic drugs. An aspartyl aminopeptidase-like gene has been identified in the Toxoplasma gondii genome ( TgAAP ), although its function remains unknown. In this study, we characterized TgAAP and performed functional analysis of the gene product. Firstly, we expressed a functional recombinant Tg AAP (r Tg AAP) protein in Escherichia coli , and found that it required metal ions for activity and showed a substrate preference for N-terminal acidic amino acids Glu and Asp. Then, we evaluated the function and drug target potential of Tg AAP using the CRISPR/Cas9 knockout system. Western blotting demonstrated the deletion of Tg AAP in the knockout strain. Indirect immunofluorescence analysis showed that Tg AAP was localized in the cytoplasm of the wild-type parasite, but was not expressed in the knockout strain. Phenotype analysis revealed that TgAAP knockout inhibited the attachment/invasion, replication, and substrate-specific activity in T. gondii . Finally, the activity of drug CID 23724194, previously described as targeting Plasmodium and malarial parasite AAP, was tested against r Tg AAP and the parasite. Overall, TgAAP knockout affected the growth of T. gondii but did not completely abolish parasite replication and growth. Therefore, Tg AAP may comprise a useful adjunct drug target of T. gondii .