The role of polyamines and related metabolites in the growth and morphology of two piscine trypanosomes
In comparison with the vast amount of literature available on the trypanosomes of mammals, relatively little is understood of those of lower vertebrates, including those present in the blood of fishes. Previous studies on fish trypanosomes have been concerned mainly with their life cycles and morphology, but this project presents data on their ultrastructure, polyamine and thiol metabolism and their responses to polyamine biosynthesis inhibitors. ‘Trypanosoma granulosum’ from the European eel and ‘Trypanosoma danilewskyi’ from the crucian carp were isolated from the blood of their respective hosts and successfully cultured ‘in vitro’ in an undefined diphasic medium. Several semi-defined and fully-defined media were then tested for their suitability to sustain growth of the parasites. A modified version of SDM-79 successfully provided the growth and, conditions necessary for morphological, ultrastructural and biochemical studies of both parasites. The ultrastructure of bloodstream ‘T. granulosum’ highlighted similarities with some stercorarian trypanosomes. In diphasic medium and modified SDM-79 the bloodstream parasites transformed into forms similar to those reportedly found in their leech vector. Mainly trypomastigote forms were present in modified SDM-79, although epimastigotes, not found in any blood smears were also present. Ultrastructurally, cultured ‘T. granulosum’ was similar to other members' of the genus ‘Trypanosoma’. Biochemical analyses of the parasites using high performance liquid chromatography, enzyme assays and radiolabelling studies revealed that their polyamine and thiol metabolisms were unusual amongst trypanosomatids for example the African trypanosomes and ‘Leishmania’ spp. Low levels of intracellular free polyamines, a rapid high affinity saturable uptake system for putrescine, the presence of glutathione as the major intracellular thiol, and the presence of the newly discovered thiol homotrypanothione, suggested that both ‘T. granulosum’ and ‘T. danilewskyi’ were similar to ‘Trypanosoma cruzi’ in their polyamine and thiol metabolism. Known inhibitors of polyamine metabolism Dl-[alpha]-difluoromethylornithine (DFMO), methylglyoxal-bis-(guanylhydrazone) (MGBG) and diminazene aceturate (Berenil) were all shown to inhibit growth and to cause drastic alterations to the morphology and ultrastructure of ‘T. granulosum’ (and ‘T. danilewskyi’). These anti-parasitic actions did not appear to result from polyamine depletion.