The lysosome-like vacuolar compartment (VAC) is a major site of proteolysis in the intracellular parasite Toxoplasma gondii Previous studies have shown that genetic ablation of a VAC-residing
cysteine protease,
cathepsin protease L (CPL), resulted in the accumulation of undigested
protein in the VAC and loss of parasite viability during the chronic stage of
infection. However, since the maturation of another VAC localizing
protease,
cathepsin protease B (CPB), is dependent on CPL, it remained unknown whether these defects result directly from ablation of CPL or indirectly from a lack of CPB maturation. Likewise, although a previously described
cathepsin D-like
aspartyl protease 1 (ASP1) could also play a role in proteolysis, its definitive residence and function in the Toxoplasma endolysosomal system were not well defined. Here, we demonstrate that CPB is not necessary for
protein turnover in the VAC and that CPB-deficient parasites have normal growth and viability in both the acute and chronic stages of
infection. We also show that ASP1 depends on CPL for correct maturation, and it resides in the T. gondii VAC, where, similar to CPB, it plays a dispensable role in protein digestion. Taken together with previous work, our findings suggest that CPL is the dominant
protease in a hierarchy of
proteolytic enzymes within the VAC. This unusual lack of redundancy for CPL in T. gondii makes it a single exploitable target for disrupting chronic
toxoplasmosis.IMPORTANCE Roughly one-third of the human population is chronically infected with the intracellular single-celled parasite Toxoplasma gondii, but little is known about how this organism persists inside people. Previous research suggested that a parasite
proteolytic enzyme, termed
cathepsin protease L, is important for Toxoplasma persistence; however, it remained possible that other associated
proteolytic enzymes could also be involved in the long-term survival of the parasite during
infection. Here, we show that two
proteolytic enzymes associated with
cathepsin protease L play dispensable roles and are dependent on
cathepsin L to reach maturity, which differs from the corresponding
enzymes in humans. These findings establish a divergent hierarchy of
proteases and help focus attention principally on
cathepsin protease L as a potential target for interrupting Toxoplasma
chronic infection.