Identification and characterization of an ectophosphatase activity involved in Acanthamoeba castellanii adhesion to host cells

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: K_m = 2.12 ± 0.54 mM p-NPP and V_max = 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.