Related Experiment Video
Updated: Jul 12, 2025
Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection
Published on: June 12, 2014
Identification and characterization of an ectophosphatase activity involved in Acanthamoeba castellanii adhesion to
Luiz Fernando Carvalho-Kelly1, Anita Leocadio Freitas-Mesquita1, Clara Ferreira Pralon1
1Instituto de Bioquímica Médica Leopoldo de Meis (IBqM), UFRJ, Rio de Janeiro, RJ, Brazil.
Abstract:
Acanthamoeba castellanii is a free-living amoeba and an opportunistic pathogen for humans that can cause encephalitis and, more commonly, Acanthamoeba keratitis. During its life cycle, A. castellanii may present as proliferative and infective trophozoites or resistant cysts. The adhesion of trophozoites to host cells is a key first step in the pathogenesis of infection. A major virulence protein of Acanthamoeba is a mannose-binding protein (MBP) that mediates the adhesion of amoebae to cell surfaces. Ectophosphatases are ecto-enzymes that can dephosphorylate extracellular substrates and have already been described in several microorganisms. Regarding their physiological roles, there is consistent evidence that ectophosphatase activities play an important role in parasite-host interactions. In the present work, we identified and biochemically characterized the ectophosphatase activity of A. castellanii. The ectophosphatase activity is acidic, stimulated by magnesium, cobalt and nickel, and presents the following apparent kinetic parameters: Km = 2.12 ± 0.54 mM p-NPP and Vmax = 26.12 ± 2.53 nmol p-NP × h-1 × 10-6 cells. We observed that sodium orthovanadate, ammonium molybdate, sodium fluoride, and inorganic phosphate are able to inhibit ectophosphatase activity. Comparing the two stages of the A. castellanii lifecycle, ectophosphatase activity is significantly higher in trophozoites than in cysts. The ectophosphatase activity is stimulated by mannose residues and is significantly increased when trophozoites interact with LLC-MK2 cells. The inhibition of ectophosphatase by pretreatment with sodium orthovanadate also inhibits the adhesion of trophozoites to epithelial cells. These results allow us to conclude that the ectophosphatase activity of A. castellanii is somehow important for the adhesion of trophozoites to their host cells. According to our data, we believe that the activation of MBP by mannose residues triggers the stimulation of ectophosphatase activity to facilitate the adhesion process.
Insights
Acanthamoeba castellanii ectophosphatase activity is higher in its infective trophozoite stage and crucial for host cell adhesion. This finding suggests a new target for combating Acanthamoeba infections.
Area of Science:
- Microbiology
- Biochemistry
- Parasitology
Background:
- Acanthamoeba castellanii is an opportunistic human pathogen causing keratitis and encephalitis.
- Trophozoite adhesion to host cells is vital for A. castellanii pathogenesis.
- Mannose-binding protein (MBP) is a key virulence factor mediating amoebal adhesion.
Purpose of the Study:
- To identify and biochemically characterize ectophosphatase activity in A. castellanii.
- To investigate the role of ectophosphatase in A. castellanii trophozoite adhesion to host cells.
Main Methods:
- Biochemical characterization of ectophosphatase activity, including kinetic analysis and inhibitor screening.
- Comparison of ectophosphatase activity between A. castellanii trophozoites and cysts.
- Assay of ectophosphatase activity during trophozoite interaction with epithelial cells (LLC-MK2).
- Inhibition of ectophosphatase activity using sodium orthovanadate to assess its effect on trophozoite adhesion.
Main Results:
- A. castellanii possesses acidic ectophosphatase activity, stimulated by Mg2+, Co2+, and Ni2+.
- Kinetic parameters (Km, Vmax) for p-NPP hydrolysis were determined.
- Ectophosphatase activity was significantly higher in trophozoites than in cysts.
- Activity was stimulated by mannose and increased upon interaction with LLC-MK2 cells.
- Inhibition of ectophosphatase by sodium orthovanadate reduced trophozoite adhesion.
Conclusions:
- Acanthamoeba castellanii ectophosphatase activity plays a significant role in trophozoite adhesion to host cells.
- Mannose-activated MBP likely stimulates ectophosphatase activity, facilitating the adhesion process.
- Ectophosphatase represents a potential therapeutic target for Acanthamoeba infections.
