Extracellular Vesicles Secreted by Acanthamoeba culbertsoni Have COX and Proteolytic Activity and Induce Hemolysis

Abstract

Several species of Acanthamoeba genus are potential pathogens and etiological agents of several diseases. The pathogenic mechanisms carried out by these amoebae in different target tissues have been documented, evidencing the relevant role of contact-dependent mechanisms. With the purpose of describing the pathogenic processes carried out by these protozoans more precisely, we considered it important to determine the emission of extracellular vesicles (EVs) as part of the contact-independent pathogenicity mechanisms of A. culbertsoni, a highly pathogenic strain. Through transmission electronic microscopy (TEM) and nanoparticle tracking analysis (NTA), EVs were characterized. EVs showed lipid membrane and a size between 60 and 855 nm. The secretion of large vesicles was corroborated by confocal and TEM microscopy. The SDS-PAGE of EVs showed proteins of 45 to 200 kDa. Antigenic recognition was determined by Western Blot, and the internalization of EVs by trophozoites was observed through Dil-labeled EVs. In addition, some EVs biological characteristics were determined, such as proteolytic, hemolytic and COX activity. Furthermore, we highlighted the presence of leishmanolysin in trophozites and EVs. These results suggest that EVs are part of a contact-independent mechanism, which, together with contact-dependent ones, allow for a better understanding of the pathogenicity carried out by Acanthamoeba culbertsoni.

Keywords: Acanthamoeba culbertsoni; COX; extracellular vesicles; pathogenicity mechanisms.

Figure 1

EVs of A. culbertsoni. (A) TEM. Representative electron micrographs of the spherical EVs delimited by a membrane. Bars = 250 nm. (B,C) NTA through the Nanosight NS300 equipment. (B) Graphic representation of the size and concentration of extracellular vesicles. The average is represented by the black line and the standard deviation by the red line. (C) Dot plot of particle size/relative intensity. Assays were performed in triplicate.

Figure 2

Representative images of the probable secretion of extracellular vesicles by A. culbertsoni. (A) Confocal microscopy. Spherical particles (arrows) were observed close to a trophozoite, which were recognized by polyclonal anti-A culbertsoni antibodies (Bar = 10 µm). (B) TEM. It was possible to observe a spherical particle with a lipid bilayer (arrowhead) close to a trophozoite (Bar = 500 nm).

Figure 3

Protein pattern and immunorecognition of A. culbertsoni extracellular vesicles. Lane 1–3: electrophoresis. Lane 1: molecular weight marker. Lane 2: extracellular vesicles. Lane 3: trophozoites. Enriched protein bands of between 45 and 200 kDa were observed in the vesicles. Lane 4: Western Blot. Antigenic recognition is observed mainly in protein bands greater than 45 kDa. Lane 5: Nonspecific binding of secondary antibody was not observed.

Figure 4

Representative images of the EVs internalization by A. culbertsoni trophozoites. EVs were isolated from a trophozoite culture, subsequently stained with DiI, and finally interacted with A. culbertsoni trophozoites for 30 min. (A,B) EVs were observed in the cytoplasm of trophozoites, suggesting intra-species communication. (C) Trophozoites in the absence of EVs were used as a control. (Bars = 10 µm).

Figure 5

Zymograms. Proteolytic activity was observed in high molecular weight areas, which is more evident in trophozoites. Lane 1: EVs. Lane 2: trophozoites.

Figure 6

Erythrocytes incubated with A. culbertsoni EVs lysis. (A) After data analysis, it was found that there was significant erythrocyte lysis in blood cells that interacted with EVs. The average ± standard deviation of the mean is presented (n = 3, *** p < 0.001). (B) Representative image of hemolysis observed.

Figure 7

Determination of COX activity in trophozoites and EVs of A. culbertsoni. Higher COX activity was observed in vesicles than in trophozoites. The average ± standard deviation of the mean of COX-like activity is presented (n = 3, * p < 0.05).

Figure 8

(A) Representative image detection of a homologous protein to leishmanolysin (leishmanolysin-like). Protein was detected in trophozoite (T). Similarly, particles that suggests EVs (arrowheads) are shown to be recognized by anti-leishmanolysin antibody, which were near the trophozoites. (B) Nonspecific binding of secondary antibody was not observed. Bars = 5 µm.