Toxoplasma gondii Cathepsin L Is the Primary Target of the Invasion-inhibitory Compound Morpholinurea-leucyl-homophenyl-vinyl Sulfone Phenyl

Abstract

The protozoan parasite Toxoplasma gondii relies on posttranslational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS), which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 Å resolution crystal structure of TgCPL in complex with its propeptide. We discuss possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion.

Solution Structure of IseA, an Inhibitor Protein of dl-Endopeptidases from Bacillus subtilis, Reveals a Novel Fold with a Characteristic Inhibitory Loop

“…Moreover, the structures of cathepsin L cysteine proteases with their propeptides from human and Toxoplasma gondii have been reported (53,54). The amino acid sequences of the propeptides bound to the active-site cleft are MNGFQ (residues 75p-79p) from human and YLGFK (residues 177p-181p) from T. gondii.…”

Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites

“…The non-vital function of TgASP1 could be explained by a redundancy between the ASPs, however this hypothesis is not very plausible based on the phylogeny and the distinct subcellular localizations of these proteases. The rhomboid like protease TgROM1 and cysteine protease TgCPL are other examples of dispensable enzymes in T. gondii for which the deletion conferred very modest effects on tachyzoites (Brossier et al, 2008;Larson et al, 2009). A more prominent role for these proteases in other life stages can be envisioned and indeed TgASP1 is expressed in both tachyzoites and bradyzoites (Shea et al, 2007).…”

Type II Toxoplasma gondiiKU80Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection

“…Nine distinct targeted gene deletions (1,6,13,32,38,54,55,57,65,69) have been successfully developed in type II strains since genetic transformation of T. gondii was reported nearly 20 years ago (18,39,61). In contrast, recently reported type I KU80 knockout strains that exhibit highly efficient gene replacement frequencies has significantly accelerated the development of targeted gene deletions (2,3,15,20,23,26,34,37,44). Increased efficiency of double-crossover homologous recombination at targeted loci in KU80 knockouts is due to the functional loss of the nonhomologous end-joining DNA repair pathway that mediates a major mechanism underlying frequent random insertion of linear episomes in T. gondii (26).…”