Proteases as Markers for Differentiation of Pathogenic and Nonpathogenic Species ofAcanthamoeba
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Fig. 1.Extracellular protease activities of pathogenic and nonpathogenic isolates of Acanthamoeba obtained by colorimetric assay. Sp1, Acanthamoeba sp.; Sp2, Acanthamoeba sp.; Sp3, A. castellanii; Sp4, Acanthamoeba sp.; Sp9,A. griffini; Sp5, A. palestinensis; Sp6,A. astronyxis. Error bar, standard deviation of the mean. One unit of enzyme activity is 1 μmol of substrate converted per min.
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Fig. 2.(A) Representative effects of intactAcanthamoeba cells on immortalized corneal epithelial cells after 12 h. P represents pathogenic and NP represents nonpathogenic species or strains. (B) Epithelial-cell monolayer disruption after 24 h using 10 or 30% ACM from Sp1 (P) or Sp6 (NP). Wells containing 30% ACM are indicated by arrows.
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Fig. 3.Different Acanthamoeba isolates exhibit different protease banding patterns.Acanthamoeba organisms (106) were incubated in 1 ml of MEM without serum at 37°C in a 5% CO2 incubator for 24 h. The parasites were removed from the medium by centrifugation (100 × g for 5 min). Five microliters of the supernatant was diluted (1:1) in sample buffer, loaded onto an SDS-PAGE gel, and electrophoresed. Following this, SDS was removed by washing in 2.5% Triton, and the gel was incubated overnight in developing buffer (pH 7.5) containing 10 mM CaCl2. Note that different isolates ofAcanthamoeba exhibit different banding patterns. Lane 1, Acanthamoeba sp. (Sp2); lane 2,Acanthamoeba sp. (Sp1); lane 3,Acanthamoeba sp. (Sp4); lane 4, A. castellanii (Sp3); lane 5, Acanthamoeba sp. (Sp5); lane 6, A. polyphaga (Sp7); lane 7, A. astronyxis (Sp6); lane 8, A. palestinensis (Sp5).
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Fig. 4.Different culture conditions alter the protease banding patterns. Acanthamoeba organisms (Sp4; 106) were incubated in 1 ml of MEM without serum under different culture conditions. Lane 1, conditioned medium fromAcanthamoeba organisms incubated with epithelial cells at 37°C in a 5% CO2 incubator; lane 2,Acanthamoeba organisms incubated without epithelial cells (ACM) at 37°C in a 5% CO2 incubator; lane 3, Acanthamoeba organisms incubated without epithelial cells (ACM) at 37°C in an atmospheric concentration of CO2; lane 4, conditioned medium fromAcanthamoeba sp. (Sp4; pathogenic) treated with 1 mM PMSF, showing complete inhibition; lane 5, ACM from A. astronyxis (nonpathogenic [Sp6]); lane 6, ACM from A. astronyxis treated with 1 mM PMSF, showing partial inhibition. Note the change in the protease banding pattern with different culture conditions. Five microliters of ACM was used per lane.
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Fig. 5.Overexpression of a 107-kDa protease in pathogenic isolates of Acanthamoeba only.Acanthamoeba parasites (106) were incubated in 1 ml of MEM without serum at 37°C at an atmospheric concentration of CO2 for 24 h. The parasites were removed from the media by centrifugation (100 × g for 5 min). Five microliters of the supernatant was diluted (1:1) in sample buffer, loaded onto an SDS-PAGE gel, and electrophoresed. Following this, SDS was removed by washing in 2.5% Triton, and the gel was incubated overnight in developing buffer (pH 7.5) containing 10 mM CaCl2. Note that the 107-kDa protease is overexpressed only in pathogenic Acanthamoeba strains. Lane 1,A. astronyxis (Sp6); lane 2, A. palestinensis(Sp5); lane 3, A. polyphaga (Sp7); lane 4,Acanthamoeba sp. (Sp4); lane 5,Acanthamoeba sp. (Sp9); lane 6, A. castellanii (Sp3); lane 7, Acanthamoeba sp. (Sp2); lane 8, Acanthamoeba sp. (Sp1).
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