Isolation of Acanthamoeba T5 from Water: Characterization of Its Pathogenic Potential, Including the Production of Extracellular Vesicles

Abstract

Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 °C, which could have implications in the parasite´s survival and damaging potential in two different biological environments.

Distinct immunomodulatory properties of extracellular vesicles released by different strains of Acanthamoeba

“…The disappearance of a 47‐kDa band also indicated the existence of cysteine and metalloproteases in the EVs cargo. These findings corroborate previous studies that showed predominance of serine proteases and the presence in lower amount of metallo‐ and cysteine proteases in Acanthamoeba , either in conditioned medium (Alsam et al, 2005; Cirelli et al, 2020; Khan, 2006; Serrano‐Luna et al, 2006; Sissons et al 2005; Ramírez‐Rico et al, 2015), or in EVs (Gonçalves et al, 2018; Retana‐Moreira et al, 2020).…”

“…In the last years, studies have demonstrated the release of EVs by pathogenic protozoan and that these vesicles can act crucially in mechanisms of invasion and host immunomodulation (Mantel & Marti, 2014; Soares et al, 2017; Torrecilhas et al, 2012; Twu et al, 2013). EVs are also released by Acanthamoeba trophozoites and their possible involvement with contact‐independent mechanism of pathogenicity was recently shown (Gonçalves et al, 2018; Lin et al, 2019; Retana‐Moreira et al, 2020).…”

“…Based on the microscopy studies (SEM and TEM), those strains successfully released EVs under the conditions of our experiments. Previous studies have shown that the sizes of Acanthamoeba EVs may range from 111.3 to 166.7 nm (Gonçalves et al, 2018; Lin et al, 2019; Retana‐Moreira et al, 2020). Consistent with those observations, Acanthamoeba ‐derived EVs exhibited a homogeneous profile, with more than 92% of particles found in the expected range from 100 to 200 nm (average size of 137.2 ± 14.7 nm).…”

“…They are important to either free‐living organisms that must feed on bacteria or to parasitic trophozoites during invasion (Khan, 2006; Magliano et al, 2009), and are also involved in Acanthamoeba encystation process, specially cysteine proteases (Wang et al, 2020). Most proteases' studies were performed in Acanthamoeba trophozoites and only recently they were found as part of EVs cargo (Gonçalves et al, 2018; Lin et al, 2019; Retana‐Moreira et al, 2020). Confirming those findings, we also detected proteases activities in the EVs from the four strains at pH 7.4 and 3.5.…”

“…Recently, EVs were described as potential virulence factors in Acanthamoeba showing protease activity and causing cytotoxicity to culture cells (Gonçalves et al, 2018; Lin et al, 2019; Retana‐Moreira et al, 2020). In other protozoans including Leishmania and Trypanosoma cruzi , EVs shed by parasitic cells are involved in the modulation of immune responses exhibiting inter and intraspecies variations (Atayde et al, 2016; Barbosa et al, 2018; Nogueira et al, 2015).…”