The role of the insulinase‐like protein Toxolysin‐2 in host cell infection by Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes severe disease in immunocompromised individuals and neonates. As the most experimentally tractable of the Apicomplexans, it also serves as a model system for studying other Apicomplexans such as Plasmodium falciparum, the causative agent of malaria. Toxoplasma's intracellular lifestyle is dependent on the ability to salvage host constituents, a process that is likely to involve parasite‐derived proteases. We are focusing on the role of secreted insulinase‐like metalloproteases in the establishment and maintenance of intracellular survival. To do this, we have engineered 6His‐tagged portions of a metalloprotease named Toxolysin‐2 for recombinant expression in E. coli. We purified the recombinant protein using nickel‐agarose chromatography for immunization of mice and antibody production. We are also directly assessing the role of the proteases using a gene knockout approach. This approach employs PCR amplification and subcloning of regions flanking the gene into a knockout construct that contains the selectable marker HXGPRT and a downstream GFP marker to distinguish between homologous and heterologous recombinants. Using this approach, we have disrupted the Toxolysin‐2 gene and are studying the effect of the knockout in vitro and in vivo. Our long‐term goal is to determine if the proteins are active metalloproteases using in vitro assays. Studying proteases is useful for the development of therapeutic treatments such as protease inhibitors, which may block cleavage events necessary for parasitic survival. Research Support: UCLA CARE Program to MP, UCLA Microbial Pathogenesis Training Grant to BEH, and NIH RO1 AI064616 to PJB