Stimulation of Acanthamoeba castellanii excystment by enzyme treatment and consequences on trophozoite growth

Abstract

Acanthamoeba castellanii is a widespread Free-Living Amoeba (FLA) that can cause severe ocular or cerebral infections in immunocompetent and immunocompromised patients, respectively, besides its capacity to transport diverse pathogens. During their life cycle, FLA can alternate between a vegetative form, called a trophozoite, and a latent and resistant form, called a cyst. This resistant form is characterized by the presence of a cell wall containing two layers, namely the ectocyst and the endocyst, mainly composed of cellulose and proteins. In the present work, we aimed to stimulate Acanthamoeba castellanii excystment by treating their cysts with a cellulolytic enzyme, i.e., cellulase, or two proteolytic enzymes, i.e., collagenase and pepsin. While 11 days were necessary to obtain total excystment in the control at 27°C, only 48 h were sufficient at the same temperature to obtain 100% trophozoites in the presence of 25 U/mL cellulase, 50 U/mL collagenase or 100 U/mL pepsin. Additionally, more than 96% amoebae have excysted after only 24 h with 7.5 U/mL cellulase at 30°C. Nevertheless, no effect of the three enzymes was observed on the excystment of Balamuthia mandrillaris and Vermamoeba vermiformis. Surprisingly, A. castellanii trophozoites excysted in the presence of cellulase displayed a markedly shorter doubling time at 7 h, in comparison to the control at 23 h. Likewise, trophozoites doubled their population in 9 h when both cellulose and cellulase were added to the medium, indicating that Acanthamoeba cyst wall degradation products promote their trophozoite proliferation. The analysis of cysts in epifluorescent microscopy using FITC-lectins and in electron microscopy revealed a disorganized endocyst and a reduction of the intercystic space area after cellulase treatment, implying that these cellular events are preliminary to trophozoite release during excystment. Further studies would be necessary to determine the signaling pathways involved during this amoebal differentiation process to identify new therapeutic targets for the development of anti-acanthamoebal drugs.

Keywords: Acanthamoeba; cellulase; excystment; free-living amoeba; protease; trophozoite growth.

FIGURE 1

Effect of cellulase on Acanthamoeba castellanii excystment. Proportions of A. castellanii trophozoites and cysts observed following 24 h (A), 48 h (B) and 72 h (C) of cellulase treatment at 27, 30 and 37°C as a function of enzyme concentration. The results correspond to the mean of four independent experiments ±SD.

FIGURE 2

Effect of collagenase on Acanthamoeba castellanii excystment. Proportions of A. castellanii trophozoites and cysts observed following 24 h (A), 48 h (B) and 72 h (C) of collagenase treatment at 27, 30 and 37°C as a function of enzyme concentration. The results correspond to the mean of four independent experiments ±SD.

FIGURE 3

Effect of pepsin on Acanthamoeba castellanii excystment. Proportions of A. castellanii trophozoites and cysts observed following 24 h (A), 48 h (B) and 72 h (C) of pepsin treatment at 27, 30 and 37°C as a function of enzyme concentration. The results correspond to the mean of four independent experiments ±SD.

FIGURE 4

Effect of enzyme combinations on Acanthamoeba castellanii excystment. Proportions of A. castellanii trophozoites and cysts observed following 8, 24 and 48 h of treatment by enzyme associations at 30°C. In these associations, all enzyme concentrations were at 5 U/mL. The results correspond to the mean of three independent experiments ±SD.

FIGURE 5

Effect of cellulase, collagenase and pepsin on the excystment of Acanthamoeba castellanii, Vermamoeba vermiformis, and Balamuthia mandrillaris. Phase contrast observation of A. castellanii, V. vermiformis, and B. mandrillaris before (Day 0) and after treatment of their cysts for 2 days at 30°C by 100 U/mL pepsin, 50 U/mL collagenase, or 7.5 U/mL cellulase. Scales bars = 20 µm.

FIGURE 6

Influence of cellulase and cellulose on A. castellanii trophozoite growth at 27°C. (A,B) Cyst (○) excystment and trophozoite (□) growth kinetics in the presence (A) or absence (B) of 7.5 U/mL cellulase. (C,D) Growth kinetics of trophozoites in the presence (□) or absence (*) of 7.5 U/mL cellulase after a passage following their excystment in the presence (C) or absence (D) of 7.5 U/mL cellulase. (E,F) Growth kinetics of non-freshly excysted, regularly passed, trophozoites in PYG medium alone (*) or PYG medium supplemented with 7.5 U/mL cellulase and cellulose at 1 g/L (□), 2.5 g/L (△) or 5 g/L (○), either directly to the trophozoites (E), or pre-incubated 24 h at 27°C before adding the amoebae (F). (G,H) Growth kinetics of regularly passed trophozoites in PYG culture medium alone (*) or PYG medium supplemented with cellulose (G) at 1 g/L (□), 2.5 g/L (△), or 5 g/L (○), or with glucose (H) at 50 mM (□), 100 mM (△), or 150 mM (○). The results correspond to the mean of three independent experiments ±SD.

FIGURE 7

Influence of cellulase treatment on carbohydrate localization in A. castellanii cysts. After a treatment in the presence or absence (control) of 7.5 U/mL cellulase at 27°C for 48 h, carbohydrates were labeled by FITC-lectins (green). Phase contrast and merge images are shown on top and at the bottom of the FITC-lectin image, respectively. Nine lectins were used in this experiment: Con A, GSL II, Jacalin, LCA, LEL, SBA, SWGA, VVL and WGA. Scale bars = 10 µm.

FIGURE 8

Analysis in transmission electron microscopy of A. castellanii cysts treated at 30°C for 48 h in the absence (A,B) or in the presence (C–G) of 5 U/mL cellulase. Treated cysts are characterized by a reduction of the intercystic space (C,D,F,G) in comparison to the control (A,B), with an endocyst either scarcely visualizable (C,D) or absent (F,G). Some treated cysts displayed a distance between endocyst and ectocyst comparable to the control, but with a disturbed endocyst and the presence of fibrillar material released within the intercystic space (E). In treated cysts, lipidic vesicles were mostly localized at the periphery of the amoeba (C,F), in opposition to the control (A). En, Endocyst; Ec, ectocyst; IS, Intercystic Space, N, Nucleus, V, Vacuole; White arrow: lipidic vesicle; Double black arrow: fibrillar material within the intercystic space. Scale bars represent 2 µm (A,F), and 1 µm (B,C,D,E,G). The intercystic space area (H) as well as the lipidic vesicle area (I) and number (J) were determined in cellulase-treated and untreated amoebae. The results correspond to the mean of 30 cells ±SEM. Three independent experiments were performed.