Cathepsin Cs and their role in survival of Toxoplasma gondii

Cathepsins Cs are key for the intracellular survival of the protozoan parasite, Toxoplasma gondii

Toxoplasma gondii is an opportunistic pathogen that causes serious disease in AIDS patients, recipient of solid organ transplants and in newborns who acquire the infection in utero. The life cycle of the parasite consists of two phases, i.e., asexual and sexual. The sexual phase takes place only in the members of felidae family (domestic and wild cats). The asexual phase can take place in any warm blooded animal. Infection in humans is acquired by ingesting oocysts. Inside the body sporozoites are released that invade macrophage of the intestines. These sporozoites are differentiated into tachyzoites which are motile and are distributed to other parts of the body through blood. In an immunocompetent host, the actively dividing tachyzoites differentiate into metabolically inactive bradyzoites, leading to life-long latent infection. Tachyzoites can invade any nuclear cell and survive intracellularly in a specialized parasitophorous vacuole in which the tachyzoites enter into bradyzoite stage.
Cysteine proteinases of the parasite play key roles in pathogenesis of the parasite and help in host invasion, parasite differentiation and intracellular survival. Cathepsin C also known as DPPI is one such cysteine protease that belongs to the papain family of cysteine proteases. The other proteases included in this family are cathepsin B, L, K, H and S. In contrast to these cathepsins, which have endopeptidase activity, cathepsin C has primarily exopeptidase activity. The authors of this paper have earlier characterized T. gondii cathepsin B and cathepsin L proteases. In the present work the authors show that T. gondii cathepsin Cs play important roles in tachyzoite growth and differentiation and may be used as drug targets in future.
This interesting paper is from the lab of Dr. Sharon L Reed, Department of Pathology and Medicine, University of California, San Diego, USA.

Expression and characterization of cDNAs encoding T. gondii cathepsins C:
The authors identified and cloned three cathepsin C genes from T. gondii.

Expression of cathepsins in tachyzoites and bradyzoites: The authors next studied the relative expression of different cathepsins (B, L, C1, C2 and C3) in tachyzoites using real time qPCR. Total RNA from in vitro cultured tachyzoites of T. gondii was isolates and transcribed into single stranded cDNA. Primers were designed to sequences of beta-tubulin, a tachzoite specific gene (SAG1), and cathepsins C1, C2, C3, B and L. The highest level of expression was shown by TgCPC1. The mRNA of TgCPC1 was 64% of the total TgCP mRNA. TgCPB was 21% and TgCPL was 7% of total CP RNA in tachyzoites. TgCPC3 mRNA was below the level of detection. The authors next performed a comparative real time PCR on the cDNA isolated from in vitro cultures tachyzoites and mature, encysted bradyzoites isolated from brains of chronically infected mice. TgCPC1, TgCPC2 and TgCPB were detected from tachyzoites but not from bradyzoites whereas TgCPL was detected in both.
These data suggest that the cathepsin genes are stage specifically down regulated.

Purification and catalytic properties of T. gondii cathepsin C:
The authors expressed cathepsin C1 and C2 in E. coli and also purified native enzymes from tachyzoites. These recombinant cathepsins and native cathepsins were run on a SDS PAGE and reacted with anti-CPC1 antibodies. Attempts to refold recombinant TgCPC1 and TgCPC2 as well as expression in Pichia were unsuccessful.
Using native enzymes, it was observed that resulting cathepsin C was 80% pure and reacted with TgCPC1 specific antibodies by Western blot analysis. No cross reaction with TgCPC2 antibody was observed. Furthermore, five synthetic dipeptide substrates were used to characterize the proteanse activity of purified TgCPC1 fractions.

Inhibitor profile of T. gondii cathepsin C and Inhibition of multiplication by cathepsin C inhibitor:
Host cells (fibroblasts) were infected with tachyzoites for 2hr and then after washing away the free parasites, they were treated with cathepsin C inhibitor. Cultures were incubated for 24hrs, fixed and stained with acridine orange and the number of parasites per vacuole was counted by fluorescence microscope. It was observed that cathepsin C inhibitor significantly reduced intracellular replication of inhibitor treated tachyzoites. The authors also tried to compared the morphology of inhibitor treated and control parasites by fluorescent and electron micrsoscopy. No abnormal morphology of parasites or fibroblasts was detected.

TgCPC1 knock-out mutant:

The authors then generated a TgCPC1 knock out through gene disruption and analyzed the phenotype of the resulting mutants. They compared the ability of both wild type and mutant parasites to invade and replicate in host cells. After twenty-four of invasion of the host cell, there were no significant differences in the number of cells infected or the average number of parasites per vacuole between the mutant TgCPC1 mutants and the wild-type parasite.


Localization of TgCPC:
TgCPC1 and 2 were found in dense granules in tachyzoites but not in rhoptries or micronemes.

TgCPC degrades proteins in the parasitophorous vacuoles of T. gondii: The results of these experiments suggest that cathepsin Cs are either directly or indirectly required to degrade proteins or oligopeptides in the vacolar space.