Polyamine synthesis and interconversion by the microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differenti ation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, p olyamine metabolism has also been studied as a potential drug target in mic roorganisms. Since little is known concerning polyamine metabolism in the m icrosporidia, we investigated it in Encephalitozoon cuniculi, a microspordi an associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy i ndicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homoge nous, consisting of pre-emergent (immature) spores which showed large array s of ribosomes near polar filament coils. Intact purified pre-emergent spor es incubated with [H-3] ornithine and methionine synthesized putrescine. sp ermidine, and spermine, while [C-14]spermine was converted to spermidine an d putrescine. Polyamine production from ornithine was inhibitable by DL-alp ha -difluoromethylornithine (DFMO) but not by DL-alpha -difluoro methylargi nine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylas e (AdoMetdc). and spermidine/spermine N'-acetyltransferase (SSAT) activitie s. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetde was putres cine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); argini ne decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic -type polyamine biosynthetic pathway and can interconvert exogenous polyami nes. Pre-emergent spores were metabolically active with respect to polyamin e synthesis and interconversion. while intact mature spores harvested from culture supernatants had little metabolic activity.