Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ϳ1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCEAPMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.

Amoebae of the genus Acanthamoeba are free-living protozoa that can cause granulomatous encephalitis and keratitis in humans. In this study, four clinical and three household dust isolates obtained in Vitória, Espírito Santo, Brazil were characterized by their morphological, genotypic, and physiological properties. All isolates belonged to group II according to Pussard and Pons' cyst morphology. Analysis of their 18S rDNA sequence identified one isolate from household dust as genotype T11 and the others six samples as genotype T4. Five T4 isolates presented a highly variable region (DF3) in 18S rDNA identical to those previously described. Physiological assays carried out with trophozoites in co-culture with bacteria or in axenic conditions showed all samples tolerated temperatures up to 37°C, regardless of culture method. One keratitis isolate grew at 42°C in co-culture with bacteria. Most isolates in co-culture survived at 1.0M, except a T11 isolate, which tolerated up to 0.5M. The isolates did not grow at 42°C and did not tolerate 0.5M and 1.0M under axenic condition. This is the first report of 18S rRNA gene genotyping applied to Acanthamoeba isolated from keratitis patients in Brazil. The results also indicated that osmo-tolerance is dependent on the culture system.

In the past years, extracellular vesicles (EVs) have become an important field of research since EVs have been found to play a central role in biological processes. In pathogens, EVs are involved in several events during the host–pathogen interaction, including invasion, immunomodulation, and pathology as well as parasite–parasite communication. In this report, we summarised the role of EVs in infections caused by viruses, bacteria, fungi, protozoa, and helminths based on the talks and discussions carried out during the International Society for Extracellular Vesicles (ISEV) workshop held in São Paulo (November, 2016), Brazil, entitled Cross-organism Communication by Extracellular Vesicles: Hosts, Microbes and Parasites.

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.

Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered. Acanthamoeba is a genus of free-living amoebas found in a variety of environments and distributed worldwide (1-3). The life cycle of the amoebas involves two cellular forms: one that is metabolically active, known as the trophozoite form, and a dormant form, called the cyst, that is also responsible for promoting resistance in adverse environments (3). In natural environments, members of Acanthamoeba spp. are hosts and reservoirs for many microorganisms, including pathogenic bacteria and yeasts (4-7). Recently, the study of members of the Acanthamoeba genus has gained increased attention since the description of them as natural hosts for viruses of the Mimiviridae family, which are among the largest and most complex viruses described to date (8). Despite the recent interest in both organisms, there are few studies focusing on the interactions between Acanthamoeba spp. and the infecting mimivirus (9, 10). Thus, the objective of this study was to analyze the interactions between Acanthamoeba spp. and Acanthamoeba polyphaga mimivirus (APMV) in response to encystment stimulation that may commonly happen in the natural environment.First, we performed a one-step growth curve analysis to compare the levels of replication of APMV at the amoebal trophozoite and cyst stages. Acanthamoeba castellanii cells (ATCC 30234) were seeded on 24-well microplates (Corning Incorporated, Corning, NY) in phosphate-amoeba saline (PAS) at 10 5 cells per well. The cells (cysts or trophozoites) were then infected with APMV at a multiplicity of infection (MOI) of 10, collected at different time points, and titrated as described before (8). All experiments were performed three times in triplicate. APMV was unable to infect cysts of A. castellanii, given that final viral titers remained close to the initial inoculum titer, revealing no viral replication (Fig. 1A). On the other hand, when trophozoites were infected, the viral progeny titer increased about 2.5 logs (500-fold) 24 h postinfection and evident cytopathic effect was observed (Fig. 1A). These results were confirmed by electron microscopy (12 h postinfection). APMV morphogenesis, including the presence of mature virions, was observed in trophozoites (Fig. 1B), while no virions could be seen inside the cysts (Fig. 1C).Next, we investigated if the stimulation of encystment affects APMV infection of amoebas. Amoebal trophozoites were infected with APMV after being incubated in Neff saline solution (Neff) to trigger encystment. Trophozoites were transferred to 24-well microplates, at 10 5 cells per we...