Date Published: October 16, 2015
Publisher: Public Library of Science
Author(s): Pierre-Mehdi Hammoudi, Damien Jacot, Christina Mueller, Manlio Di Cristina, Sunil Kumar Dogga, Jean-Baptiste Marq, Julia Romano, Nicolò Tosetti, Juan Dubrot, Yalin Emre, Matteo Lunghi, Isabelle Coppens, Masahiro Yamamoto, Daniel Sojka, Paco Pino, Dominique Soldati-Favre, Laura J Knoll.
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 remodelling. 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 GRAs 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. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signalling pathways and immune responses.
The phylum Apicomplexa groups obligate protozoan parasites that are the causative agents of severe diseases in humans and animals such as malaria, toxoplasmosis, babesiosis and coccidiosis. The key process of invasion and subsequent multiplication within their host cells is maintained via secretion from three distinct phylum-specific organelles termed the micronemes, rhoptries and dense granules [1–3].
Whilst intracellular, apicomplexan parasites reside within a specialised membranous niche (PVM), across which the parasite transports a plethora of effector molecules necessary for subversion and remodelling of host cell functions. Studies in P. falciparum have revealed that this process relies upon a PVM-resident translocation machinery (PTEX) that serves to facilitate export of parasite proteins across this membrane into the erythrocyte cytosol [38, 39]. Intimately associated with this process is Plasmepsin V, a protease known to cleave a specialised motif (PEXEL) in a wide repertoire of known exported proteins [40–42]. Similarly, T. gondii possesses components related to the PTEX translocon that are proposed to act as a molecular sieve at the PVM, allowing diffusion of small molecules across this membrane .