Cloning and Expression of a Molecule with Immunomodulatory Potential, a Cysteine Protease Inhibitor from Toxocara canis

Justyna Karabowicz; Oliwia Śniadała; Julia Wiśniewska; Ewa Długosz

Abstract

Parasites are known for their ability to escape from the immune system of their hosts. One of the strategies is the release of specific molecules with high immunomodulatory activity. Cysteine protease inhibitors (cystatins) have been shown to play an essential role in suppressing the host’s immune response. Cystatins have been found in many species of parasites from different taxa. Herein, we report cloning and expression of the cysteine protease inhibitor, Tc-CPI-2, from dog roundworm (Toxocara canis) infective L3 larvae, a causative agent of human toxocarosis. Sequence analysis revealed two specific cystatin-like domains, the Q-x-V-x-G motif and the S-N-D motif. Phylogenetic analysis indicated that Tc-CPI-2 belongs to type 2 cystatin subfamily. The recombinant cystatin was expressed in Escherichia coli, purified, and used to stimulate human THP-1 macrophages in vitro and mouse splenocytes ex vivo. Cell treatment with the recombinant molecule induced significant cytokine secretion changes, suggesting that it may be responsible for the Th2/Th1 immune response switch in T. canis-infected mice. Altogether, our results prove that Tc-CPI-2 is a molecule with promising immunoregulatory and therapeutic potential.

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