Final Activity Report Summary - FISHIMRES (Functional characterisation of the fish immune response to parasite) Trypanoplasma borreli and Trypanosoma carassii are extracellular parasites (Metakinetoplastida) that are transmitted by a leech vector and are naturally found in the blood of cyprinid fish. High parasitaemia and associated severe anaemia and splenomegaly are typical of infection of common carp, Cyprinus carpio. Studies on infections with kinetoplastid parasites have brought much understanding of host-parasite interactions. At present, emerging insecticide resistance in vectors and drug resistance in parasites have led to a renewed research interest in these infections models. Papain-like cysteine proteases expressed by trypanosome parasites are considered an important target for the development of new trypanocidal drugs. We demonstrated the presence of a cysteine protease expressed by both T. borreli (Parabodonida) and T. carasssii (Trypanosomatida). Alignment of these sequences with other kinetoplastid cystein protease sequences supports the phylogenetic hypotheses based on analyses of ribosomal RNA genes. We expressed the cysteine protease from both parasites in E. coli and refolded the purified protein(s) into a biologically active protease with cathepsin-L activity for T. borreli. Biologically active protein, however, could not be produced for T. carassii. Immunisation of carp with protease of T. borreli induced an increase in specific antibodies but did not lower parasitaemia. Activation of carp head kidney-derived macrophages with the protease induced an innate- rather than an alternatively-activated profile with increased amounts of nitric oxide, also typical of T. borreli infection in vivo. Gene expression of macrophages stimulated with biologically active recombinant T.borreli cysteine proteinase induced an up-regulation of IL-1ß and iNOS, confirming classical activation of macrophages. The cysteine protease shows the ability to digest host haemoglobin, immunoglobulin and transferrin, contributing to the pathogenicity of T. borreli. Our findings support a conservation of function with respect to cysteine protease activity in the Parabodonida. Further, the findings show a clear analogy of activation of the immune system of fish with that of higher vertebrates infected with Metakinetoplastidan parasites.
