Fractionation of sporogonial stages of the microsporidian. Encephalitozooncuniculi by Percoll (R) gradients

Microsporidia are obligate intracellular parasites that are increasingly re cognized as a cause of opportunistic infections in immunocompromised indivi duals. Encephalitozoon cuniculi has been identified in humans with AIDS and infects a wide range of mammalian hosts. Little is known about the metabol ic processes that regulate growth and replication of microsporidia. Examina tion of the individual stages of development will facilitate such studies a nd reveal possible targets for drug therapy. The purpose of this study was to fractionate and purify stages of the microsporidian life cycle. Encephal itozoon cuniculi were cultured in RK-13 cells. The tissue supernatants cont aining multiple parasite stages, empty microsporidial husks and host cell d ebris were collected, washed, and subjected to differential centrifugation in 80% stock isotonic Percoll(R). Transmission electron microscopy and SDS- polyacrylamide gel electrophoresis were used to compare the content and pur ity of each fraction. Mature spores formed a band at a density of approxima tely 1.138 g/ml. Sporoblasts were found at densities between 1.102 g/ml and 1.119 g/ml. A mixture of sporonts, sporoblasts, microsporidial husks, and cell debris remained at the top of the gradient and additional centrifugati on in 30% and 50% Percoll(R) resulted in separation of these stages. These results represent the first step toward fractionating stages of microsporid ia infecting humans.