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Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The effect of Camellia sinensis (green tea) on the growth of Acanthamoeba castellanii trophozoites was examined using a microplate based-Sulforhodamine B (SRB) assay. C. sinensis hot and cold brews at 75% and 100% concentrations significantly inhibited the growth of trophozoites. We also examined the structural alterations in C. sinensis-treated trophozoites using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). This analysis showed that C. sinensis compromised the cell membrane integrity and caused progressive destruction of trophozoites. C. sinensis also significantly inhibited the parasite's ability to form cysts in a dose-dependent manner and reduced the rate of excystation from cysts to trophozoites. C. sinensis exhibited low cytotoxic effects on primary corneal stromal cells. However, cytotoxicity was more pronounced in SV40-immortalized corneal epithelial cells. Chromatographic analysis showed that both hot and cold C. sinensis brews contained the same number and type of chemical compounds. This work demonstrated that C. sinensis has anti-acanthamoebic activity against trophozoite and cystic forms of A. castellanii. Further studies are warranted to identify the exact substances in C. sinensis that have the most potent anti-acanthamoebic effect., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interests., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Pathogenic protozoans use extracellular vesicles (EVs) for intercellular communication and host manipulation. Acanthamoeba castellanii is a free-living protozoan that may cause severe keratitis and fatal granulomatous encephalitis. Although several secreted molecules have been shown to play crucial roles in the pathogenesis of Acanthamoeba, the functions and components of parasite-derived EVs are far from understood., Methods: Purified EVs from A. castellanii were confirmed by electron microscopy and nanoparticle tracking analysis. The functional roles of parasite-derived EVs in the cytotoxicity to and immune response of host cells were examined. The protein composition in EVs from A. castellanii was identified and quantified by LC-MS/MS analysis., Results: EVs from A. castellanii fused with rat glioma C6 cells. The parasite-derived EVs induced an immune response from human THP-1 cells and a cytotoxic effect in C6 cells. Quantitative proteomic analysis identified a total of 130 proteins in EVs. Among the identified proteins, hydrolases (50.2%) and oxidoreductases (31.7%) were the largest protein families in EVs. Furthermore, aminopeptidase activities were confirmed in EVs from A. castellanii., Conclusions: The proteomic profiling and functional characterization of EVs from A. castellanii provide an in-depth understanding of the molecules packaged into EVs and their potential mechanisms mediating the pathogenesis of this parasite.

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals., (© 2019 John Wiley & Sons Ltd.)

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ∆ 8,14 -sterol (<95%). The combination of [ 2 H 3 -methyl]methionine incubation to intact cultures showing C 28 -ergosterol incorporates 2- 2 H atoms and C 29 -7-dehydroporiferasterol incorporates 5 2 H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ∆ 24(28) - and ∆ 25(27) -olefin pathways to C 28 - and C 29 -sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C 28 /C 29 -∆ 5,7 -sterols, viable encysted cells (mature cyst) possess mostly C 29 -∆ 5 -sterol and non-viable encysted cells possess C 28 /C 29 -∆ 5,7 -sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ∆ 5,7 -sterol biosynthesis (to ∆ 5,7 ) - metabolism (to ∆ 5 or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the host's osmoregulatory organelle. In this study, we decipher the molecular mechanisms involved in the maintenance of V. cholerae's intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrate that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, have a key role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.

Purpose: Acanthamoeba keratitis is a well-known intractable corneal infectious disease. We investigated the anti-Acanthamoeba effect of exogenous nitric oxide (NO)., Methods: Acanthamoeba castellanii was axenically cultured and exposed to various concentrations of NO donors, such as sodium nitrite, sodium nitroprusside (SNP), and NO-releasing silica nanoparticles (coated in branched polyethylene imine, size:100 nm), for 1 to 7 days (sodium nitrite and SNP: 0, 0.1, 1, 10, 100, and 1000 μM; silica nanoparticles: 0, 6.25, 12.5, 25, 50, and 100 μg/mL). Human corneal epithelial cells (HCECs) were cultured and exposed to sodium nitrite, SNP (0, 0.1, 1, 10, 100, and 1000 μM), and silica nanoparticles for 1, 2, and 3 days., Results: Sodium nitrite and SNP showed a dose-dependent inhibitory effect on A. castellanii viability. A more prominent inhibitory effect was observed with SNP (less than 10% of organisms survived at 7-day culture with 1000 μM) compared with sodium nitrite. However, more cytotoxicity on HCEC was observed with SNP. NO-releasing silica nanoparticles were successfully internalized into the amoebic cytoplasm and accumulated in large vacuoles. Although blank silica nanoparticles had no inhibitory effect on A. castellanii viability, NO-releasing silica nanoparticles showed a dose-dependent amoebicidal effect. Furthermore, no cystic transformation of A. castellanii was observed under a phase contrast microscope or transmission electron microscope after exogenous NO treatment., Conclusions: Our results demonstrated the anti-Acanthamoeba effect of exogenous NO. This finding suggests that NO-releasing drug platforms, including nano-carriers, can be a promising therapeutic strategy for Acanthamoeba keratitis.

Background: The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation., Methods: Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry., Results: Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane., Conclusions: The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out.

Bacterial infections have remained significant despite our advances in the development of a plethora of disinfectants as well as antimicrobial chemotherapy. This is in part due to our incomplete understanding of the prevalence of bacterial pathogens in the environmental and clinical settings. Several lines of evidence suggest that Acanthamoeba is one of the most ubiquitous/resilient protists that also acts as a host/reservoir for pathogenic microbes. Thus targeting the hardy host, which harbour microbial pathogens, offer a potential avenue to counter infection transmission, particularly hospital/community-acquired infections. This will complement existing approach of applying disinfectants that are targeted against bacterial pathogens directly., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus, A. castellanii is described as a Trojan horse able to harbor pathogenic bacteria. L. pneumophila is one of the amoeba-resisting bacteria able to avoid host degradation by phagocytosis and to multiply inside the amoeba. When infecting its host, L. pneumophila injects hundreds of effectors via a type IV secretion system that change physiology of the amoeba to its profit. In this study, we assess mobility of A. castellanii upon infection with L. pneumophila. Electron-microscopy analysis of amoebae revealed a reduction of acanthopodia on cells infected with L. pneumophila. Analysis of velocity showed that migration of A. castellanii infected with L. pneumophila was significantly impaired compare to uninfected cells. Taken together, infection with L. pneumophila could prevent formation of cytoplasmic extensions such as acanthopodia with consequences on the shape, adherence and mobility of A. castellanii., (Copyright © 2017 Elsevier Inc. All rights reserved.)

African swine fever virus , a double-stranded DNA virus that infects pigs, is the only known member of the Asfarviridae family. Nevertheless, during our isolation and sequencing of the complete genome of faustovirus, followed by the description of kaumoebavirus, carried out over the past 2 years, we observed the emergence of previously unknown related viruses within this group of viruses. Here we describe the isolation of pacmanvirus, a fourth member in this group, which is capable of infecting Acanthamoeba castellanii Pacmanvirus A23 has a linear compact genome of 395,405 bp, with a 33.62% G+C content. The pacmanvirus genome harbors 465 genes, with a high coding density. An analysis of reciprocal best hits shows that 31 genes are conserved between African swine fever virus , pacmanvirus, faustovirus, and kaumoebavirus. Moreover, the major capsid protein locus of pacmanvirus appears to be different from those of kaumoebavirus and faustovirus. Overall, comparative and genomic analyses reveal the emergence of a new group or cluster of viruses encompassing African swine fever virus , faustovirus, pacmanvirus, and kaumoebavirus. IMPORTANCE Pacmanvirus is a newly discovered icosahedral double-stranded DNA virus that was isolated from an environmental sample by amoeba coculture. We describe herein its structure and replicative cycle, along with genomic analysis and genomic comparisons with previously known viruses. This virus represents the third virus, after faustovirus and kaumoebavirus, that is most closely related to classical representatives of the Asfarviridae family. These results highlight the emergence of previously unknown double-stranded DNA viruses which delineate and extend the diversity of a group around the asfarvirus members., (Copyright © 2017 American Society for Microbiology.)

Nontuberculous mycobacteria (NTM) are environmental bacteria increasingly associated to public health problems. In water systems, free-living amoebae (FLA) feed on bacteria by phagocytosis, but several bacteria, including many NTM, are resistant to this predation. Thus, FLA can be seen as a training ground for pathogenic bacteria. Mycobacterium llatzerense was previously described as frequently associated with FLA in a drinking water network. The present study aimed to characterize the interactions between M. llatzerense and FLA. M. llatzerense was internalised by phagocytosis and featured lipid inclusions, suggesting a subversion of host resources. Moreover, M. llatzerense survived and even multiplied in presence of A. castellanii. Using a genomic-based comparative approach, twelve genes involved in phagocytosis interference, described in M. tuberculosis, were identified in the M. llatzerense genome sequenced in this study. Transcriptomic analyses showed that ten genes were significantly upregulated during the first hours of the infection, which could partly explain M. llatzerense resistance. Additionally, M. llatzerense was shown to actively inhibit phagosome acidification. In conclusion, M. llatzerense presents a high degree of resistance to phagocytosis, likely explaining its frequent occurrence within FLA in drinking water networks. It underscores that NTM should be carefully monitored in water networks to prevent human health concerns.

The survival of three Arcobacter butzleri strains inside Acanthamoeba castellanii was assessed using axenic cultures of A. castellanii that were inoculated with the tested strains and incubated at 26°C under aerobic conditions for 240h. The behavior of bacteria in contact with amoebae was monitored using phase contrast microscopy. The bacterial survival rate within amoebae was assessed through counting colony forming units, using the gentamicin protection assay. All A. butzleri strains were able to survive during 240h within the amoebae, thus suggesting that (i) A. butzleri resists the amoebic digestion processes at least for the analyzed time; (ii) that A. castellanii could serve as an environmental reservoir for this bacterium, probably acting as a transmission vehicle for A. butzleri., (Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Autophagy is a well conserved, catabolic process in eukaryotic cells. Previously, we identified two novel ubiquitin like conjugation systems (Atg12 and Atg8) in the autophagy process of Acanthamoeba castellanii. To obtain more specific information on the Atg12 ubiquitin like conjugation system during encystation of Acanthamoeba, we characterized the function of Atg12. Knockdown of AcAg12 in trophozoites resulted in inhibition of cyst formation. Analysis of subcellular localization showed that AcAtg12 was evenly distributed in the trophozoites during early encystation, started to accumulate partially as dots or fragments, and then co-localized with the vesicle of the autophagic structure. However, the mRNA expression of AcAtg12 was maintained at a constant level during encystation as well as in trophozoites. Ultrastructural analysis with TEM showed that AcAtg12-knockdown cells showed vacuolization, lack of cyst wall formation, and numerical decline of autophagic structures, compared with the control cells. Interestingly, these knockdown cells began to round-up and swell, and then burst at 144 h post encystation. Taken together, our results might provide a better understanding of the Atg12 UBL conjugation system in Acanthamoeba and other cyst forming protozoan parasites., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available. Transmission electron microscopy (TEM) is ideally suited for those analyses; however, conventional TEM protocols are not satisfactory for cysts of free-living protozoa. Here we report on the design and testing of four protocols for TEM sample preparation of cysts. Two protocols, one based on chemical fixation in coated well plates and one on high-pressure freezing, were selected as the most effective for TEM-based ultrastructural studies of cysts. Our protocols will enable improved analysis of cyst structure and a better understanding of bacterial survival mechanisms in cysts.

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens., (© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.)

Painful blinding keratitis and fatal granulomatous amebic encephalitis are caused by the free-living amebae Acanthamoeba spp. Several prescription eye medications are used to treat Acanthamoeba keratitis, but the infection can be difficult to control because of recurrence of infection. For the treatment of encephalitis, no single drug was found useful, and in spite of the use of a combination of multiple drugs, the mortality rate remains high. Therefore, efficient, novel drugs are urgently needed for the treatment of amebic keratitis and granulomatous amebic encephalitis. In this study, we identified corifungin, a water-soluble polyene macrolide, as amebicidal. In vitro, it was effective against both the trophozoites and the cysts. Transmission electron microscopy of Acanthamoeba castellanii incubated with corifungin showed the presence of swollen mitochondria, electron-dense granules, degeneration of cytoplasm architecture, and loss of nuclear chromatin structure. These changes were followed by lysis of amebae. Corifungin also induced the encystment process of A. castellanii. There were alterations in the cyst cell wall followed by lysis of the cysts. Corifungin is a promising therapeutic option for keratitis and granulomatous amebic encephalitis.

In this study, Acanthamoeba castellanii was cultivated under different stress conditions to induce possible encystation. The morphological and histological properties were analysed by light and electron microscopy as well as cyst-specific staining. The findings revealed that cysts prepared through liquid medium using higher osmolarity as a trigger (10% glucose with 50mM magnesium chloride for 72 h) are similar to cysts prepared using non-nutrient agar (nutrient deprivation as a trigger in plating assays for 14 days), as determined by SDS-resistance, cyst-specific Calcofluor white staining and transmission electron microscopy. Using liquid medium assay, A. castellanii encystation was studied by exposing trophozoites to media lacking growth ingredients (phosphate buffered saline or distilled water), inappropriate temperatures (4-45°C), pH (3-9), artificial light-dark cycles, 5% CO2, and microaerophilic conditions. Optimal encystation was observed when cells were incubated in PBS with 50mM MgCl2 and 10% glucose at 24-30°C at pH 7. Increasing temperature over 37°C or pH 9 adversely affected encystation, while light-dark cycles, 5% CO2 and microaerophilic conditions had no effect on encystation of A. castellanii. None of the aforementioned conditions had any effect on the viability of A. castellanii, as determined by Trypan blue exclusion assay. A complete knowledge of encystation in A. castellanii is crucial to our understanding of the biology of these ecologically and medically important organisms., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mitochondria are the result of a billion years of integrative evolution, converting a once free-living bacterium to an organelle deeply linked to diverse cellular processes. One way in which mitochondria are integrated with nonendosymbiotically derived organelles is via endoplasmic reticulum (ER)-mitochondria contact sites. The ER membrane is physically tethered to the mitochondrial outer membrane by the ER-mitochondria encounter structure (ERMES). However, to date, ERMES has only ever been found in the fungal lineage. Here, we bioinformatically demonstrate that ERMES is present in lineages outside Fungi and validate this inference by mass spectrometric identification of ERMES components in Acanthamoeba castellanii mitochondria. We further demonstrate that ERMES is retained in hydrogenosome-bearing but not mitosome-bearing organisms, yielding insight into the process of reductive mitochondrial evolution. Finally, we find that the taxonomic distribution of ERMES is most consistent with rooting the eukaryotic tree between Amorphea (Animals + Fungi + Amoebozoa) + Excavata and all other eukaryotes (Diaphoratickes).

During Acanthamoeba castellanii trophozoite-cysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst, immature cysts, and mature cysts. Fluorescence microscopy reveals the presence of small cumulus of actin in the cytoplasm of precysts after treatment with rhodamine phalloidin. By the contrary, in mature cysts, fluorescence was not observed. However, when excystation was induced, large fluorescent patches were present. By transmission electron microscopy, encysting amebas showed small cytoplasmic vesicles containing fibrillar material, surrounded by a narrow area of thin fibrils. Similar appearance was observed in pseudopods and phagocytic invaginations. In addition, large aggregates of rod-shape elements, similar to the chromatoid bodies, described in other amebas, were present in the cytoplasm. These cysts presented large areas with orange fluorescence after treatment with acridine orange.

Enterohemorrhagic Escherichia coli (EHEC) are involved in outbreaks of food-borne illness and transmitted to humans through bovine products or water contaminated by cattle feces. Microbial interaction is one of the strategies used by pathogenic bacteria to survive in the environment. Among protozoa, the free-living amoebae are known to host and protect several water-borne pathogens. In this study, the interaction between EHEC and the predacious protozoa Acanthamoeba castellanii was investigated. Using monoculture and cocultures, growth of both organisms was estimated for 3 weeks by total and viable cell counts. The numbers of EHEC were significantly higher when cultured with amoebae than without, and less EHEC shifted into a viable but nonculturable state in the presence of amoebae. Using several mutants, we observed that the Pho regulon is required for EHEC growth when cocultured with amoebae. In contrast, the Shiga toxins (Stx) were not involved in this association phenotype. Cocultures monitored by electron microscopy revealed a loss of the regular rod shape of EHEC and the secretion of multilamellar vesicles by the amoebae, which did not contain bacteria. As the interaction between A. castellanii and EHEC appears beneficial for bacterial growth, this supports a potential role for protozoa in promoting the persistence of EHEC in the environment., (© 2012 The Authors. Published by Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Crude methanol extracts of a marine sponge, Aaptos aaptos, collected from three different localities namely Kapas, Perhentian and Redang Islands, Terengganu, Malaysia, were tested in vitro on a pathogenic Acanthamoeba castellanii (IMR isolate) to examine their anti-amoebic potential. The examination of anti-Acanthamoebic activity of the extracts was conducted in 24 well plates for 72 h at 30 °C. All extracts possessed anti-amoebic activity with their IC(50) values ranging from 0.615 to 0.876 mg/mL. The effect of the methanol extracts on the surface morphology of A. castellanii was analysed under scanning electron microscopy. The ability of the extracts to disrupt the amoeba cell membrane was indicated by extensive cell's blebbing, changes in the surface morphology, reduced in cell size and with cystic appearance of extract-treated Acanthamoeba. Number of acanthapodia and food cup was also reduced in this Acanthamoeba. Morphological criteria of apoptosis in Acanthamoeba following treatment with the sponge's extracts was determined by acridine orange-propidium iodide staining and observed by fluorescence microscopy. By this technique, apoptotic and necrotic cells can be visualized and quantified. The genotoxic potential of the methanol extracts was performed by the alkaline comet assay. All methanol extracts used were significantly induced DNA damage compared to untreated Acanthamoeba by having high percentage of scores 1, 2, and 3 of the DNA damage. Results from cytotoxicity and genotoxicity studies carried out in the present study suggest that all methanol extracts of A. aaptos have anti-amoebic properties against A. castellanii.

Chlorination is a well-known disinfection method, used in water treatment to inactivate various microorganisms, it induces numerous cellular changes. Even though Acanthamoebae are frequently found in water, the cellular changes induced in Acanthamoebae have not been described in the literature. Acanthamoebae are pathogenic amoebae and may provide a reservoir for pathogenic bacteria such as Legionellapneumophila; it is consequently important to understand the response of this amoeba to chlorination, and our study was indeed aimed at examining cellular changes in Acanthamoebae following chlorination. Acanthamoeba trophozoites were treated at various chlorine concentrations (1-5mg/L). A 3-log reduction in Acanthamoebae population was achieved with 5mg/L of free chlorine. Confocal microscopy and flow cytometry experiments indicated that chlorination induced cell permeabilization, size reduction and likely intracellular thiol concentration. Our data show that among the non-cultivable cells some remained impermeabilized (negative staining with propidium iodide), thereby suggesting that these cells might remained viable. A similar state is described in other microorganisms as a VBNC (viable but not cultivable) state. Electron microscopy observations illustrate drastic morphological changes: the pseudopods disappeared and subcellular components, such as mitochondrion, were pronouncedly affected. In conclusion, depending on the concentration used, chlorination leads to many cellular effects on Acanthamoeba that could well arise in cell inactivation., (Copyright 2009 Elsevier Inc. All rights reserved.)

The present study demonstrates that when Acanthamoeba castellanii trophozoites are co-cultivated with isolated human corneas, the amoeba can be invasive and cause damage to the intact corneal epithelium without the requirement of previous corneal abrasion. After adhesion, A. castellanii trophozoites migrate between cells forming bumps on the corneal cell layers and reaching Bowman s membrane in 3h, although no evidence of cell damage was observed until the phagocytic process was detected. Likewise, conditioned medium produced damage to the corneal cells that was proportional to the time of incubation, but this cytophatic effect involved only the most superficial layer of the human cornea and was not enough to explain amoebic invasion of Bowman s membrane. As a result of our observations, we suggest that the mechanical action of the trophozoites and phagocytosis of corneal cells during the process of corneal invasion are more important than previously suggested., (Copyright 2010 Elsevier Inc. All rights reserved.)

We analyzed biochemically Acanthamoeba castellanii trophozoites, intact cysts and cyst walls belonging to the T4 genotype using gas chromatography combined with mass spectrometry. Cyst walls were prepared by removing intracellular material from cysts by pre-treating them with sodium dodecyl sulphate (SDS) containing dithiothreitol, and then subjecting these to a series of sequential enzymatic digestions using amyloglucosidase, papain, DNase, RNase and proteinase K. The resulting "cyst wall" material was subsequently lyophilized and subjected to glycosyl composition analysis. Transmission electron microscopy confirmed the removal of intracystic material following enzymatic treatment. Our results showed that treated A. castellanii trophozoites, intact cysts and cyst walls contained various sugar moieties, of which a high percentage was galactose and glucose, in addition to small amounts of mannose, and xylose. Linkage analysis revealed several types of glycosidic linkages including the 1,4-linked glucosyl conformation, indicative of cellulose. Inhibitor studies suggested that, beside sugar synthesis, cytoskeletal re-arrangement and mitogen-activated protein kinase-mediated pathways are involved in A. castellanii encystment.

Legionella pneumophila is known as a facultative intracellular parasite of free-living soil and freshwater amoebae, of which several species have been shown to support the growth of the pathogenic bacteria. We report for the first time the behaviour of two strains (c2c and Z503) of the amoeba Willaertia magna towards different strains of L. pneumophila serogroup 1 and compared it with Acanthamoeba castellanii and Hartmannella vermiformis, known to be L. pneumophila permissive. In contrast to the results seen with other amoebae, W. magna c2c inhibited the growth of one strain of Legionella (L. pneumophila, Paris), but not of others belonging to the same serogroup (L. pneumophila, Philadelphia and L. pneumophila, Lens). Also, the different L. pneumophila inhibited cell growth and induced cell death in A. castellanii, H. vermiformis and W. magna Z503 within 3-4 days while W. magna c2c strain remained unaffected even up to 7 days. Electron microscopy demonstrated that the formation of numerous replicative phagosomes observed within Acanthamoeba and Hartmannella is rarely seen in W. magna c2c cocultured with L. pneumophila. Moreover, the morphological differences were observed between L. pneumophila cultured either with Willaertia or other amoebae. These observations show that amoebae are not all equally permissive to L. pneumophila and highlight W. magna c2c as particularly resistant towards some strains of this bacterium.

Vibrio cholerae, the causative agent of cholera, has the ability to grow and survive in the aquatic free-living amoeba Acanthamoeba castellanii. The aim of the present study was to examine the ability of the clinical isolate V. cholerae O139 MO10 to grow in A. castellanii and to determine the effect of the bacterial capsule and LPS O side chain on intracellular growth. Results from co-cultivation, viable counts, a gentamicin assay, electron microscopy and statistical analysis showed that the association of V. cholerae O139 MO10 with A. castellanii did not inhibit growth of the amoeba, and enhanced growth and survival of V. cholerae O139 MO10 occurred. The wild-type V. cholerae O139 MO10 and a capsule mutant or capsule/LPS double mutant grew inside A. castellanii. Neither the capsule nor the LPS O side chain of V. cholerae O139 was found to play an important role in the interaction with A. castellanii, disclosing the ability of V. cholerae to multiply and survive inside A. castellanii, as well as the role of A. castellanii as an environmental host for V. cholerae.

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparison between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.

Objectives: Acanthamoeba keratitis (AK) is most commonly found in contact lens wearers with poor lens care hygiene. Recent reports have indicated an increase in cases of AK in the United States associated with the use of Complete MoisturePlus multipurpose solution. This association may not appear explainable by differences in Acanthamoeba kill rates among multipurpose solutions. In this study the physiologic response of Acanthamoeba trophozoites to incubation in various contact lens solutions was investigated., Methods: Acanthamoeba trophozoites were incubated in commercial and experimental solutions and the percentage encystment and viability determined in conjunction with microscopic analysis of cyst formation., Results: Among commercial products, MoisturePlus solution induced encystment within 6 to 24 hr of incubation. Only immature cysts formed which failed to mature even after 5 days incubation in the solution although they remained viable. Experimental formulations of MoisturePlus solution, modified by omitting key ingredients, identified propylene glycol as the agent responsible for inducing encystment. However, tests of other commercial solutions containing propylene glycol produced reduced or no encystment., Conclusions: The presence of propylene glycol was shown to be the key factor in inducing Acanthamoeba encystment. However, the reduced or absence of encystment in other commercial solutions containing propylene glycol suggests additional factors, such the buffering system, may be involved in the phenomenon. How these observations relate to the solution's role in the reported association with AK is unclear. Nevertheless, it is recommended that all lens care products be tested for propensity to induce Acanthamoeba encystment and formulations containing propylene glycol may warrant special scrutiny.

Pseudomonas aeruginosa is a free-living and common environmental bacterium. It is an opportunistic and nosocomial pathogen causing serious human health problems. To overcome its predators, such as macrophages and environmental phagocytes, it utilises different survival strategies, such as the formation of microcolonies and the production of toxins mediated by a type III secretion system (TTSS). The aim of this study was to examine interaction of TTSS effector proteins of P. aeruginosa PA103 with Acanthamoeba castellanii by co-cultivation, viable count, eosin staining, electron microscopy, apoptosis assay, and statistical analysis. The results showed that P. aeruginosa PA103 induced necrosis and apoptosis to kill A. castellanii by the effects of TTSS effector proteins ExoU, ExoS, ExoT, and ExoY. In comparison, Acanthamoeba cultured alone and co-cultured with P. aeruginosa PA103 lacking the known four TTSS effector proteins were not killed. The results are consistent with P. aeruginosa being a strict extracellular bacterium that needs TTSS to survive in the environment, because the TTSS effector proteins are able to kill its eukaryotic predators, such as Acanthamoeba.

Acanthamoeba castellanii (T4) was cultured with different concentrations of allitridium for 24 hours, and examined by transmission electron microscope. The results showed that the ultrastructure of Acanthamoeba trophozoites was destroyed gently at concentration of 50 microg/ml allitridium and seriously destroyed under the concentration of 500 microg/ml, indicating that allitridium is effective in destroying Acanthamoeba.

Purpose: To evaluate the cysticidal effect of polyhexamethylene biguanide (PHMB) and chlorhexidine on Acanthamoeba and its toxic effect on cultured human keratocytes., Methods: Each well of a twofold-diluted Acanthamoeba cyst-containing suspension of 5 x 10(4) cysts/mL was treated with PHMB and chlorhexidine for 8, 24, and 48 hours to determine the minimal cysticidal concentration (MCC) of each disinfectant. Human corneal keratocytes (5 x 10(4) cells/mL) were exposed to PHMB and chlorhexidine for the same time to determine the survival rate of keratocytes. Inverted phase-contrast and electron microscopy were used to observe the morphologic changes., Results: The mean MCC of PHMB for 8, 24, and 48 hours was 9.42, 5.62, and 2.37 microg/mL, respectively. The mean MCC of chlorhexidine for 8, 24, and 48 hours was 24.32, 10.02, and 7.02 microg/mL, respectively. The respective survival rate of keratocytes at the MCC was 91.7%, 64.6%, and 49.7% for PHMB and 95.7%, 90.6%, and 78.1% for chlorhexidine, respectively. The cysts and keratocytes showed more damaged appearances after treatment with PHMB than chlorhexidine., Conclusions: PHMB and chlorhexidine showed a similar amoebicidal efficacy. However, PHMB seemed to be more toxic to keratocytes than chlorhexidine.

Transmission electron microscopy (TEM) can provide high resolution imaging of biological specimens. The study is to establish the effects of a modified glutaraldehyde (GA) compare to the standard GA fixation on Acanthamoeba castellanii from TEM perspectives and thus provide precise and accurate information on the ultrastructure studies of the parasite. By increasing the contrast, the ultrastructures of the parasite were more evident. The TEM images were obtained from parasites fixed with the modified GA and the standard GA and then the area of the nucleus and the gray values of the image of the nucleus of the parasites were measured. The mean areas of the nucleus were found to be significantly reduced in the standard GA fixed parasites (12210.4 nm2) compared to the modified GA fixed parasites (8676.3 nm2) (p < 0.05). The mean gray values of the image were significantly reduced from 2024 in the standard GA fixed parasites (2024) to the modified GA fixed parasites (1636) (p < 0.05). The study shows that the modified GA produced significantly better contrast on TEM images of the A. castellanii compared to the standard GA. This was because the modified GA generated more free water molecules during fixation and the uptake of modified GA by the nucleus of the parasite organizing all protein constituents in the cell into a more closely packed configuration than that of the standard GA. With such properties, the modified GA is a better fixative providing better images for ultrastructures of the parasite.

It is suggested that in the course of the TOM complex evolution at least two lineages have appeared: the animal-fungal and green plant ones. The latter involves also the TOM complexes of algae and protozoans. The amoeba Acanthamoeba castellanii is a free-living non-photosynthetic soil protozoan, whose mitochondria share many bioenergetic properties with mitochondria of plants, animals and fungi. Here, we report that a protein complex, identified electrophysiologically as the A. castellanii TOM complex, contains a homologue of yeast/animal Tom 70. Further, molecular weight of the complex (about 500 kDa) also points to A. castellanii evolutionary relation with fungi and animal. Thus, the data indicates that the TOM complex of A. castellanii is not a typical example of the protozoan TOM complex.

Vibrio cholerae is a highly infectious bacterium responsible for large outbreaks of cholera among humans at regular intervals. A seasonal distribution of epidemics is known but the role of naturally occurring habitats are virtually unknown. Plankton has been suggested to play a role, because bacteria can attach to such organisms forming a biofilm. Acanthamoebea castellanii is an environmental amoeba that has been shown to be able to ingest and promote growth of several bacteria of different origin. The aim of the present study was to determine whether or not an intra-amoebic behaviour of V. cholerae O139 exists. Interaction between these microorganisms in co-culture was studied by culturable counts, gentamicin assay, electron microscopy, and polymerase chain reaction. The interaction resulted in intra-amoebic growth and survival of V. cholerae in the cytoplasm of trophozoites as well as in the cysts of A. castellanii. These data show symbiosis between these microorganisms, a facultative intracellular behaviour of V. cholerae contradicting the generally held view, and a role of free-living amoebae as hosts for V. cholerae O139. Taken together, this opens new doors to study the ecology, immunity, epidemiology, and treatment of cholera.

In Acanthamoeba castellanii mitochondria, the apparent affinity values of alternative oxidase for oxygen were much lower than those for cytochrome c oxidase. For unstimulated alternative oxidase, the K(Mox) values were around 4-5 microM both in mitochondria oxidizing 1 mM external NADH or 10 mM succinate. For alternative oxidase fully stimulated by 1 mM GMP, the KK(Mox) values were markedly different when compared to those in the absence of GMP and they varied when different respiratory substrates were oxidized (K(Mox) was around 1.2 microM for succinate and around 11 microM for NADH). Thus, with succinate as a reducing substrate, the activation of alternative oxidase (with GMP) resulted in the oxidation of the ubiquinone pool, and a corresponding decrease in K(Mox). However, when external NADH was oxidized, the ubiquinone pool was further reduced (albeit slightly) with alternative oxidase activation, and the K(Mox) increased dramatically. Thus, the apparent affinity of alternative oxidase for oxygen decreased when the ubiquinone reduction level increased either by changing the activator or the respiratory substrate availability.

Encystation and excystation of Acanthamoeba castellanii were studied by transmission electron microscopy. The differentiation process was induced in asynchronous cultures grown axenically. Cytoplasmic vesicles containing a dense fibrous material very similar in appearance to the cyst wall were observed in trophozoites induced to encyst. When these trophozoites were incubated with calcofluor white m2r, fluorescence was observed in cytoplasmic vesicles, suggesting that the material contained in these vesicles corresponded to cyst wall precursors. Semithin cryosections of mature cysts with the same treatment showed fluorescence in the ectocyst and a less intense fluorescence in the endocyst, suggesting the presence of cellulose in both structures of the cyst wall. In mature cysts induced to excystation, small structures very similar to electron-dense granules (EDG) previously described in other amoebae were frequently observed. The EDGs were either sparsely distributed in the cytoplasm or associated with the cytoplasmic face of the plasma membrane. Many of them were located near the ostiole. In advanced phases of excystation, endocytic activity was suggested by the formation of endocytic structures and the presence of vacuoles with fibrous content similar to that of the cyst wall. Electron-dense granules in the process of dissolution were also observed in these vacuoles. Furthermore, the formation of a pseudopod suggests a displacement of the amoeba toward the ostiole.