PARTIAL CHARACTERIZATION OF THE PROTEOLYTIC SECRETIONS OF ACANTHAMOEBA-POLYPHAGA

The ocular pathology associated with acanthamoebiasis may result, at l east in part, from the excretory and secretory (EBLS) products of the active Acantharnoeba trophozoites. To test this hypothesis, the abilit y of A. polyphaga (ATCC Strain 30461) trophozoite E&S products to dige st collagen, the major constitutent of the corneal stroma, was evaluat ed. The secreted proteinases of A, polyphaga were identified using in vitro azocoll degradation, activity-PAGE, radiolabeled extracellular m atrix (ECM) degradation, and collagen degradation assays. Inhibitors o f serine (phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate), cysteine [benzytoxyphenylalanyl-analyl fluoromethyl ketone, N-ethylma leamide, ethylenediamine tetraacetic acid (EDTA), -trans-3-carboxyiran -2-carbonyl-L-leucylagmatine], metallo-(1,10-phenanthroline, EDTA, pho sphoramidon), and aspartyl (pepstatin A) proteinases were incorporated into the assays. Proteinase activity was detected in trophozoites and the E&S products of trophozoites but not in cysts. The azocoll and ac tivity-PAGE assays indicated the presence of serine and cysteine prote inases, while degradation of the radiolabeled ECM by live trophozoites confirmed not only the presence of serine and cysteine proteinases bu t also metalloproteinase(s). Most proteinase activity occurred at neut ral pH. Incubation of E&S with type I collagen did not yield the typic al 3/4:1/4 products characteristic of vertebrate collagenases. These d ata suggest that E&S products of A. polyphaga contain multiple serine and cysteine proteinases with nonspecific collagenolytic activity and that metallproteinases form an additional minor constituent. (C) 1994 Academic Press, inc,