Partial Characterization of the Proteolytic Secretions of Acanthamoeba polyphaga

doi:10.1006/expr.1994.1041

Experimental Parasitology

Volume 78, Issue 4, June 1994, Pages 377-385

https://doi.org/10.1006/expr.1994.1041 Get rights and content

Abstract

The ocular pathology associated with acanthamoebiasis may result, at least in part, from the excretory and secretory (E&S) products of the active Acantharnoeba trophozoites. To test this hypothesis, the ability of A. polyphaga (ATCC Strain 30461) trophozoite E&S products to digest collagen, the major constitutent 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 [benzytoxyphenylalanyl-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 $34$ : $14$ 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.

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Supernatants were collected, and their absorbances were read with a spectrophotometer at 520 nm to determine the Azocoll activities as reported by Serrano-Luna et al, (2006) [25]. Protease activities were reported as milliunits per milligram; this unit of measure is equivalent to the amount of substrate degraded in 1 min per milligram of TCE or per milliliter of CM [9]. TCE and CM were preincubated with protease inhibitors (at the concentrations described above) at room temperature for 1 h before incubation with the substrate.
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Tissue damage and invasion then proceed through phagocytic activity and cytopathic effects by molecules released by the parasites; among them are proteolytic enzymes (Alizadeh et al., 1994; Mattana et al., 2002; Marciano-Cabral and Cabral, 2003; Alsam et al., 2005; Clarke and Niederkorn, 2006). In addition to studies demonstrating that secretion of certain serine and metallopeptidases can be induced by trophozoite–host cell contact or addition of mannose to the culture medium (Cao et al., 1998; Leher et al., 1998; Hurt et al., 2003), Acanthamoeba spp. constitutively secrete appreciable amounts of serine and cysteine peptidases (He et al., 1990; Mitro et al., 1994; Mitra et al., 1995; Alfieri et al., 2000; Kong et al., 2000; Cho et al., 2000; Na et al., 2001; Sissons et al., 2006). Constitutive secretion of metallopeptidases has also been demonstrated in A. castellanii (Alsam et al., 2005; Sissons et al., 2006), and suggested in Acanthamoeba polyphaga (Mitro et al., 1994; Alfieri et al., 2000).
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Regular ArticlePartial Characterization of the Proteolytic Secretions of Acanthamoeba polyphaga

Abstract

Regular Article
Partial Characterization of the Proteolytic Secretions of Acanthamoeba polyphaga