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

Abstract

Free living amoeba of the genus Acanthamoeba are opportunist protozoan involved in corneal, systemic, and encephalic infections in humans. Most of the mechanisms underlying intraspecies variations and pathogenicity are still unknown. Recently, the release of extracellular vesicles (EVs) by Acanthamoeba was reported. However, comparative characterization of EVs from distinct strains is not available. The aim of this study was to evaluate EVs produced by Acanthamoeba from different genotypes, comparing their proteases profile and immunomodulatory properties. EVs from four environmental or clinical strains (genotypes T1, T2, T4, and T11) were obtained by ultracentrifugation, quantitated by nanoparticle tracking analysis and analyzed by scanning and transmission electron microscopy. Proteases profile was determined by zymography and functional properties of EVs (measure of nitrite and cytokine production) were determined after peritoneal macrophage stimulation. Despite their genotype, all strains released EVs and no differences in size and/or concentration were detected. EVs exhibited a predominant activity of serine proteases (pH 7.4 and 3.5), with higher intensity in T4 and T1 strains. EVs from the environmental, nonpathogenic T11 strain exhibited a more proinflammatory profile, inducing higher levels of Nitrite, tumor necrosis factor alpha and interleukin‐6 via TLR4/TLR2 than those strains with pathogenic traits (T4, T1, and T2). Preincubation with EVs treated with protease inhibitors or heating drastically decreased nitrite concentration production in macrophages. Those data suggest that immunomodulatory effects of EVs may reflect their pathogenic potential depending on the Acanthamoeba strains and are dependent on protease integrity.

Characterization of Extracellular Vesicles Secreted by a Clinical Isolate of Naegleria fowleri and Identification of Immunogenic Components within Their Protein Cargo

“…A complete and partial inhibition of protease activity was achieved when incubating EVs with phenylmethylsulfonyl fluoride and EDTA, respectively. These results suggest that the predominant enzymatic activity in EVs is related to serine proteases, followed by a less intense metalloprotease activity (Figure 4), similar to that described for clinical and environmental isolates of Acanthamoeba [25,26,28,46]. In contrast, published reports of pro-tease activity of whole protein extracts of N. fowleri trophozoites suggest a predominance of cysteine proteases [63,66].…”

“…Besides this immunomodulatory activity, EVs could act as vehicles for virulence factors like the major surface protease (MSP), also known as GP63 [ 58 , 59 ]. Regarding free-living amoebae, Costa et al have demonstrated that EVs of clinical and environmental isolates of Acanthamoeba are capable of inducing the production of nitric oxide via TLR4 in an in vitro model using murine macrophages [ 28 ]. These authors concluded that EVs carry different types of PAMPs that could trigger the innate immune response during the infection process.…”

“…More recently, and in the case of free-living amoebae with pathogenic potential, the secretion of EVs has been described in Acanthamoeba , a genus related to the production of fatal granulomatous amoebic encephalitis, keratitis and skin lesions. In this specific case, biological and nanomechanical properties of extracellular vesicles produced by clinical and environmental isolates of Acanthamoeba have been recently reported [ 25 , 26 , 27 , 28 ].…”

Three encephalitis-causing amoebae and their distinct interactions with the host

“…TNF-α, IL-6, and IL-12 are regarded as proinflammatory cytokines, but the significant production of the anti-inflammatory cytokine IL-10 suggests a mixed-type immune response which might allow immune evasion of Acanthamoeba and limit the inflammatory response [66]. Furthermore, pathogenic Acanthamoeba strains induce a mixed-type immune response, whereas nonpathogenic Acanthamoeba strains induce mainly a proinflammatory response [67,68], which could result in rapid elimination of nonpathogenic Acanthamoeba spp.…”

The Role of Extracellular Vesicles from Human Macrophages on Host-Pathogen Interaction

“…Many studies have shown multiple ways by which several pathogens modify EV synthesis, enhancing or restricting their replication and dissemination in receptor cells [ 11 , 12 , 13 ]. In fact, there is accumulating evidence of the release of vesicles from several sources during infectious diseases, acting both in pathogen–pathogen inter-communication as well as in host–pathogen interaction [ 11 , 14 , 15 , 16 , 17 , 18 , 19 ]. However, despite the increasing number of reviews describing and discussing the involvement and role of EVs in the complex scenario of host–pathogen interaction, up-to-date information about the specific impact of vesicles released by human macrophages on the progression of infectious illnesses is quite limited.…”