| Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, we have identified a target enzyme class, the aminopeptidases and investigated the biochemical properties within several species of microsporidia. Possible aminopeptidase inhibitors were investigated in vitro using a rabbit kidney endothelial cell line infected with Encephalitozoon cuniculi. Lead compounds were further evaluated in an athymic mouse model infected with E. cuniculi.; Aminopeptidase activity was detected in 4 species of microsporidia; Encephalitozoon intestinalis, Encephalitozoon cuniculi, Encephalitozoon hellem and Vittaforma corneae using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon sPP. were characterized as preferentially cleaving leucine, while V. corneae cleaved arginine. The aminopeptidase activity identified in each species was sensitive to bestatin.; Two in vitro assays were used to evaluate the efficacy and toxicity of compounds against E. cuniculi in a mammalian cell line. The classic aminopeptidase inhibitor, bestatin and its analogues (amastatin and nitrobestatin) and the controversial aminopeptidase inhibitors, fumagillin and TNP-470 were tested for their effects on the replication of E. cuniculi in culture. All 5 drugs showed dose dependent inhibitory activity. Host cell toxicity as measured by the MTT assay, indicated minimal toxic effects with bestatin and analogues. However, host cell viability was decreased with fumagillin and TNP-470.; An in vivo model was utilized to investigate the effects of TNP-470 and bestatin on the survival time of athymic mice infected with E. cuniculi . TNP-470 was observed to increase the survival time of mice. However, the activity was not dose dependent. Bestatin prolonged the survival time of infected mice and the action was both route of administration and dose dependent.; This study presents initial evidence that microsporidial aminopeptidases may be regarded as an important target to which therapeutic compounds could be directed. This study also indicates that bestatin is a possible lead compound for therapeutic control of microsporidia. |
