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Sensitivity of Enzymatic Toxins from Corneal Isolate of Acanthamoeba Protozoan to Physicochemical Parameters
Viviane P Sant'Ana1, Annette S Foronda1, Denise de Freitas1
1Department of Ophthalmology and Visual Sciences, Paulista School of Medicine, Federal University of Sao Paulo, Botucatu Street, 821, Vila Clementino, Sao Paulo, SP, 04023-062, Brazil.
Abstract:
Acanthamoeba is a free-living amoeba that causes severe corneal infection (Acanthamoeba keratitis) and produces a variety of extracellular enzymes, called exoproteome. Since physicochemical characters are suggested being associated with therapeutic profile and clinical severity of the infection, we investigated the physicochemical properties of proteolysis mediated by amoebic exoproteome. Corneal scraping was collected from a patient who showed typical symptoms of acute Acanthamoeba keratitis. Axenic amoeba was phylogenetically identified by 18S rDNA sequencing analysis. Effects of pH, temperature and diamidines on proteolysis mediated by exoproteome were assessed using zymography assays. Proteolytic enzymes were most active at pH 7.0 and 37 °C. Calcium ions decreased enzymatic activity. The main components of amoebic exoproteome were characterized as serine proteases. We demonstrated for the first time that commercial antimicrobial diamidines used for Acanthamoeba keratitis therapy inhibit enzymatic activity of amoebic exoproteome. Results showed the thermostability of Acanthamoeba proteases, which suggest a long-term effect of these virulence factors at the central and peripheral cornea with possible role in degradation of extracellular matrix components. Our findings open new perspectives about the complementary and unreported properties of antimicrobial compounds of the diamidine class on the inhibition of enzymatic activity and presumptive control of amoebic infection in the cornea.
Insights
Acanthamoeba keratitis virulence factors, Acanthamoeba proteases, are most active at pH 7.0 and 37°C. Antimicrobial diamidines inhibit these enzymes, offering new therapeutic strategies for corneal infections.
Area of Science:
- Microbiology
- Ophthalmology
- Biochemistry
Background:
- Acanthamoeba causes severe corneal infections (Acanthamoeba keratitis).
- Acanthamoeba exoproteome contains extracellular enzymes implicated in infection severity.
- Physicochemical properties of these enzymes may influence therapeutic outcomes.
Purpose of the Study:
- To investigate the physicochemical properties of proteolysis mediated by Acanthamoeba exoproteome.
- To determine the optimal conditions for Acanthamoeba protease activity.
- To assess the effect of diamidines on protease activity.
Main Methods:
- Corneal scraping from an Acanthamoeba keratitis patient.
- Phylogenetic identification of Acanthamoeba using 18S rDNA sequencing.
- Zymography assays to assess protease activity under varying pH, temperature, and in the presence of calcium ions and diamidines.
Main Results:
- Acanthamoeba proteases exhibited optimal activity at pH 7.0 and 37°C.
- Calcium ions reduced enzymatic activity.
- The primary exoproteome components were identified as serine proteases.
- Commercial antimicrobial diamidines significantly inhibited protease activity.
Conclusions:
- Acanthamoeba proteases are thermostable, suggesting prolonged virulence factor activity in the cornea.
- Antimicrobial diamidines demonstrate a novel inhibitory effect on Acanthamoeba exoproteome activity.
- These findings suggest a complementary role for diamidines in controlling Acanthamoeba keratitis by inhibiting key enzymes.
