Molecular cloning, recombinant expression and characterisation of serine and cysteine protease inhibitors from Trichinella zimbabwensis.

Abstract

Trichinellosis is a disease caused by parasitic helminths of the genus Trichinella. Infection occurs by ingestion of meat contaminated with infective Trichinella larvae. Cases of Trichinella zimbabwensis, a non-encapsulating species of Trichinella infecting mammals and reptiles, have been reported in Ethiopia, Mozambique and South Africa. The parasite life cycle alternates between the enteric and skeletal muscle phases of infection. Trichinella species release various excretory-secretory products that enable successful parasitism. These include cysteine protease inhibitors, cystatins and serine protease inhibitors. To illustrate, cystatins have roles in cellular invasion and immune evasion while serpins inhibit blood coagulation, resist host protease damage and interfere with host immunoregulatory signals. The potential roles of endogenous parasite cysteine and serine protease inhibitors in T. zimbabwensis make these inhibitors attractive targets for the development of novel antiparasitic interventions. The genes encoding a cysteine protease inhibitor, cystatin B, and a Kazal-type serine protease inhibitor, SPINK4, were identified in the T. zimbabwensis genome. Following the synthesis of cDNA from nematode extracted mRNA, the respective genes were amplified and cloned into E. coli expression vectors. The recombinantly expressed proteins, rTzcystatin B and rTzSPINK4, were purified using immobilised metal affinity chromatography, their inhibitory activity evaluated and antibodies were produced in chickens. The antibodies were used for the detection of the recombinant proteins on western blots and ELISA. Recombinant Tzcystatin B inhibited the activity of the catalytic domain of the cathepsin L-like peptidase from Trypanosoma congolense (TcoCATL), the homologues from T. vivax (TviCATL) and Theileria parva (ThpCATL) as well as cathepsin B from T. zimbabwensis (TzCATB). Conversely, rTzSPINK4 was unable to inhibit either host serine proteases, chymotrypsin or trypsin. Future studies will be aimed at exploring the effect of the T. zimbabwensis protease inhibitors on host proteases involved in antigen processing. This may indicate a possible role for the nematode protease inhibitors in host immunoregulation.