RNA-Seq analysis of mouse BMDMs infected RH and Pru strain Asp5-KO parasites. TgAsp5KO-BMDMs Transcriptome

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodeling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRA proteins fail to localize to the PVM, while GRA16 and GRA24 (both known to be targeted to the host cell nucleus), are retained within the vacuolar space. RNA-Seq analysis on mouse bone marrow derived macrophages (BMDMs) infected with ASP5-KO parasites has been done to identify the differentially expressed genes in the host cell, particularly those pertaining to immune responses and signalling pathways. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signaling pathways and immune responses. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence.