Aspartyl Protease 5 matures virulence factors found at the host-parasite interface in Toxoplasma gondii

Toxoplasma gondii infects approximately 30% of the worlds population, causing disease primarily during pregnancy and in individuals with weakened immune systems. Toxoplasma secretes and exports effector proteins that modulate the host during infection and several of these proteins are processed by the Golgi-associated Aspartyl Protease 5 (ASP5). Here, we identify ASP5 substrates by selectively enriching N-terminally-derived peptides from wildtype and {Delta}asp5 parasites. We reveal over two thousand unique Toxoplasma N-terminal peptides, mapping to both natural N-termini and protease cleavage sites. Several of these peptides mapped directly downstream of the characterised ASP5-cleavage site, arginine-arginine-leucine (RRL). We validate candidates as true ASP5 substrates, revealing they are not processed in parasites lacking ASP5, nor in wild type parasites following mutation of the motif from RRL{longrightarrow}ARL. All new ASP5 substrates are dense granule proteins, and interestingly none appear to be exported, thus differing from the analogous system in related Plasmodium spp., instead revealing that the majority of substrates reside within the parasitophorous vacuole (PV), and its membrane (the PVM), including two kinases and one phosphatase. Furthermore, we show that several of these ASP5-substrates are virulence factors, with their removal leading to attenuation in a mouse model, suggesting that phosphorylation at the host-parasite interface is important for virulence. Collectively, these data constitute the first in-depth analyses of the total list of ASP5 substrates, and shed new light on the role of ASP5 as a maturase of dense granule proteins during the Toxoplasma lytic cycle.