Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote- containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.