CPL

TgCPL is a cysteine protease that acts as the dominant endolysosomal protease of the Toxoplasma gondii plant-like vacuolar compartment (PLVAC/VAC), where it drives proteolytic maturation and bulk protein digestion required for nutrient scavenging and chronic-stage viability (PMID:32051238, PMID:40172222). It sits at the top of a VAC proteolytic hierarchy, being required for maturation of the downstream proteases CPB and ASP1, such that its ablation—but not that of CPB or ASP1—causes accumulation of undigested protein and loss of viability during chronic infection (PMID:32051238). CPL degrades host-derived cytosolic macromolecules internalized by both tachyzoites and bradyzoites; in its absence host-expressed reporter protein accumulates within the endolysosomal compartment, and Δcpl parasites show reduced amino acids and glycolytic intermediates and exacerbated growth defects under amino acid depletion, establishing its role in supplying nutrients from the host (PMID:33504659, PMID:40172222). Its proteolytic maturation depends on acidification of the VAC, supported redundantly by TgVP1 or the V-H+-ATPase (PMID:24975633). CPL is the primary molecular target of the invasion-inhibitory compound LHVS, and its active site has been validated as a druggable target: a 2.0 Å crystal structure of the CPL–propeptide complex enabled structure-based design of dipeptidic inhibitors with ~50-fold selectivity over human cathepsin L (PMID:19596863, PMID:29650289).

1. 2009 High

   Establishing what LHVS actually inhibits was needed to interpret its anti-invasion activity; identifying CPL as the selective target and solving its structure gave a molecular handle on the enzyme.

   Evidence 2.0 Å crystal structure of CPL–propeptide complex, fluorescent LHVS labeling, and CPL gene disruption in two strains

   PMID:19596863

   Open questions at the time

   • Cellular substrates not yet defined
   • Functional consequence of specific subcellular localization not established
2. 2009 Medium

   Locating CPL within the parasite was needed to place its activity in a cellular compartment; labeling showed apical vesicular and cytoplasmic punctate distributions.

   Evidence Fluorescent LHVS labeling and CPL-specific antibody immunolocalization

   PMID:19596863

   Open questions at the time

   • Localization not yet functionally linked to any activity
   • Compartment identity not defined at this stage
3. 2013 Low

   Whether CPL processes secretory cargo was addressed by associating it with maturation of proTgM2AP and proTgMIC3 and with the residual body.

   Evidence Claims cited from prior experimental work in a review/vaccine-study context

   PMID:23651838

   Open questions at the time

   • Cited secondary source without independent experimental validation here
   • Direct enzyme–substrate relationship not demonstrated in this work
4. 2014 Medium

   How CPL becomes active was unresolved; showing that its processing requires VAC acidification supported redundantly by TgVP1 or the V-H+-ATPase defined the conditions for its maturation.

   Evidence TgVP1-null mutant combined with bafilomycin A1 inhibition and western blot for CPL processing

   PMID:24975633

   Open questions at the time

   • Molecular mechanism of pH-dependent activation not defined
   • Autocatalytic vs trans-processing not distinguished
5. 2020 High

   The relationship among VAC proteases was unknown; reciprocal knockouts placed CPL at the apex of a proteolytic hierarchy, maturing CPB and ASP1 and being uniquely required for protein turnover and chronic viability.

   Evidence Genetic ablation of CPB and ASP1, VAC protein turnover assays, and viability assays across infection stages

   PMID:32051238

   Open questions at the time

   • Direct cleavage of CPB/ASP1 by CPL not shown biochemically in isolation
   • Contribution of each downstream protease to specific substrates unresolved
6. 2021 High

   Whether CPL digests host-derived material was tested by tracking an ingested host reporter; its accumulation in CPL-deficient parasites established CPL as required for degradation of ingested host proteins.

   Evidence CPL ablation with host-expressed mCherry accumulation assay in two cystogenic strains (ME49, Pru)

   PMID:33504659

   Open questions at the time

   • Full spectrum of host substrates not defined
   • Delivery route of host cargo to the VAC not fully mapped here
7. 2025 High

   The metabolic purpose of host-protein digestion was unclear; combining a genome-wide fitness screen, metabolomics, and amino-acid-depletion growth assays showed CPL supplies amino acids and glycolytic intermediates from the host.

   Evidence Genome-wide CRISPR fitness screen (Δcpl vs WT), bulk metabolomics, and growth in amino acid-depleted media

   PMID:40172222

   Open questions at the time

   • Individual nutrient dependencies not dissected
   • Coupling between ingestion pathway and CPL activity not mechanistically resolved
8. 2025 Medium

   Defining the CPL compartment's protein composition was addressed using CPL as a proximity-labeling bait, confirming its PLVAC residence and identifying novel PLVAC-associated proteins.

   Evidence TurboID proximity labeling with CPL bait and mass spectrometry

   PMID:39864696

   Open questions at the time

   • Functional roles of identified PLVAC proteins not established
   • Direct interactions vs mere proximity not distinguished

• The direct enzymatic substrate repertoire of CPL and the mechanism coupling host-cargo delivery to its proteolytic activity remain to be fully defined.
• No comprehensive substrate map
• Mechanism linking ingestion pathway to CPL-mediated digestion not resolved