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Partial characterization of the proteolytic secretions of Acanthamoeba polyphaga
K Mitro1, A Bhagavathiammai, O M Zhou
1Department of Ophthalmology, Medical University of South Carolina, Charleston 29425.
Abstract:
The ocular pathology associated with acanthamoebiasis may result, at least in part, from the excretory and secretory (E&S) products of the active Acanthamoeba trophozoites. To test this hypothesis, the ability of A. polyphaga (ATCC Strain 30461) trophozoite E&S products to digest collagen, the major constituent of the corneal stroma, was evaluated. The secreted proteinases of A. polyphaga were identified using in vitro azocoll degradation, activity-PAGE, radiolabeled extracellular matrix (ECM) degradation, and collagen degradation assays. Inhibitors of serine (phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate), cysteine [benzyloxyphenylalanyl-analyl fluoromethyl ketone, N-ethylmaleamide, ethylenediamine tetraacetic acid (EDTA), L-trans-3-carboxyiran-2-carbonyl-L-leucylagmatine], metallo- (1,10-phenanthroline, EDTA, phosphoramidon), 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 activity-PAGE assays indicated the presence of serine and cysteine proteinases, while degradation of the radiolabeled ECM by live trophozoites confirmed not only the presence of serine and cysteine proteinases but also metalloproteinase(s). Most proteinase activity occurred at neutral pH. Incubation of E&S with type I collagen did not yield the typical 3/4:1/4 products characteristic of vertebrate collagenases. These data 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.
Insights
Acanthamoeba excretory products contain proteinases that degrade collagen, potentially causing eye damage in acanthamoebiasis. These findings highlight serine, cysteine, and metalloproteinases in Acanthamoeba pathogenesis.
Area of Science:
- Ocular Microbiology
- Parasitology
- Biochemistry
Background:
- Ocular pathology in acanthamoebiasis is linked to Acanthamoeba excretory and secretory (E&S) products.
- Collagen is a primary component of the corneal stroma, making it a target for parasitic enzymes.
Purpose of the Study:
- To investigate the collagen-digesting capabilities of E&S products from Acanthamoeba polyphaga (ATCC Strain 30461).
- To identify the types of proteinases present in Acanthamoeba E&S products and their role in collagen degradation.
Main Methods:
- Utilized in vitro azocoll degradation, activity-PAGE, and radiolabeled extracellular matrix (ECM) degradation assays.
- Employed specific inhibitors to identify serine, cysteine, metallo-, and aspartyl proteinases.
- Assessed collagen degradation by incubating E&S products with type I collagen.
Main Results:
- Proteinase activity was detected in Acanthamoeba trophozoites and their E&S products, but not in cysts.
- Serine and cysteine proteinases were identified through azocoll and activity-PAGE assays.
- Degradation of radiolabeled ECM confirmed serine, cysteine, and metalloproteinases, with activity optimal at neutral pH.
- E&S products exhibited non-specific collagenolytic activity, differing from typical vertebrate collagenases.
Conclusions:
- Acanthamoeba E&S products contain multiple serine and cysteine proteinases with broad collagenolytic activity.
- Metalloproteinases are present as a minor component contributing to collagen degradation.
- These findings support the hypothesis that Acanthamoeba E&S products contribute to ocular pathology in acanthamoebiasis.