Biological characterization of a clinical and an environmental isolate of <Emphasis Type="Italic">Acanthamoeba polyphaga</Emphasis>: analysis of relevant parameters to decode pathogenicity

Acanthamoeba spp. consists of free-living amoebae, widespread in nature, which occasionally can cause human infections including granulomatous amoebic encephalitis and amoebic keratitis. Acanthamoeba pathogenesis is not entirely known and correlations between pathogenic potential and taxonomy are complex issues. In order to decipher the definition of a pathogenic amoeba, the objective of this work was to decipher the definition of pathogenic amoeba by characterizing two isolates of Acanthamoeba polyphaga obtained from different origins (a keratitis patient and freshwater), looking for differences among them. The clinical isolate grew faster in Peptone-yeast extract-glucose (PYG) medium, transformed more rapidly from a trophozoite to cyst and exhibited increased cytopathic effect on cultured cells. Morphological differences were also noted, since freshwater amoebae presented more acanthopodia than the clinical isolate. Moreover, actin labeling demonstrated that microfilament organization varies between isolates, with the presence of locomotory structures as lobopodia and lamellipodia in the keratitis isolate, which were less adherent on plastic. Zymography demonstrated that the keratitis isolates presented higher proteolytic activity and also were more able to invade collagen matrices. Altogether, we conclude that a group of stable physiological characteristics exist in Acanthamoeba that can be related to pathogenicity.     

Killing the Dead: Chemotherapeutic Strategies Against Free‐Living Cyst‐Forming Protists (Acanthamoeba sp. and Balamuthia mandrillaris)

The opportunist free‐living protists such as Acanthamoeba spp. and Balamuthia mandrillaris have become a serious threat to human life. As most available drugs target functional aspects of pathogens, the ability of free‐living protists to transform into metabolically inactive cyst forms presents a challenge in treatment. It is hoped, that the development of broad spectrum antiprotist agents acting against multiple cyst‐forming protists to provide target‐directed inhibition will offer a viable drug strategy in the treatment of these rare infections. Here, we present a comprehensive report on upcoming drug targets, with emphasis on cyst wall biosynthesis along with the related biochemistry of encystment pathways, as we strive to bring ourselves a step closer to being able to combat these deadly diseases.     

Short-Cut Pathway to Synthesize Cellulose of Encysting Acanthamoeba

The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.     

Effects of 2,6-dichlorobenzonitrile and Tinopal LPW on the structure of the cellulose synthesizing complexes ofVaucheria hamata

Summary The effects of 2,6-dichlorobenzonitrile (DCB, a known inhibitor of cellulose synthesis) and Tinopal LPW (TPL, an agent which disrupts glucan crystallization) on the structure of cellulose synthesizing complexes (terminal complexes, TCs) in the xanthophycean algaVaucheria hamata were investigated. DCB (10 M) inhibits nascent fibril formation from the TC subunit (based on the absence of impressions) although it does not alter the overall shape of the rectangular TC during the short treatment of 20 min. With a prolonged treatment (60 min), the arrangement of TC subunits becomes disordered, and particles generally exhibited as doublets of subunits are released from each other. DCB also interferes with the formation of the overall shape of the TC although it does not disturb the conversion into TC rows of the subunits (the zymogenic precursor of the TC) packed in the globules. A 15 min treatment with TPL (1 mM) destroys the TC integrity by reducing the subunits into small fragments or particulate aggregates. The particulate rows of the TC are interrupted at many points, and fragments and particulate aggregates are dispersed by prolonged treatment (45 min) with TPL. Unlike DCB, TPL inhibits the conversion of globule subunits into TC rows. New insights on the structural characteristics necessary for cellulose microfibril assembly and possible mechanisms for the biogenesis of theVaucheria TC come from these data.Abbreviations DCB 2,6-dichlorobenzonitrile - TPL Tinopal LPW - TC terminal complex     

Labeled Trichoderma reesei Cellulase as a Marker for Acanthamoeba Cyst Wall Cellulose in Infected Tissues

Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.Laboratory diagnosis of infections with Acanthamoeba spp. is based on identification of the parasite in infected tissue. Although various techniques, including immunocytological and molecular methods, have been described, recovery of viable parasites by cultivation on agar is still the basic procedure used (16). This method is usually associated with histopathological examination of the specimen to prove tissue invasion by the parasite.Recognition of parasites in tissue sections is often difficult and depends on the expertise of the pathologist. In addition to traditional histological staining methods, immunohistology using parasite-specific antibodies, lectin conjugates, and calcofluor white have been used for visualization of parasites in tissue sections (3).Some protozoan parasites have the ability to protect themselves by forming a cyst wall, which is resistant to environmental stresses such as desiccation, lack of nutrients, and variations in temperature and pH. In most pathogenic protozoans studied, chitin is the carbohydrate polymer providing the required structural toughness to the cyst wall. Acanthamoeba spp. are exceptions, as their cysts are made up of cellulose. Recently, cellulose has also been identified as a cyst wall component in a closely related amoeba, Balamuthia mandrillaris (15). Cellulose consists of β-d-glucosyl units linked by β-1,4-glucosidic bonds. Chitin is very similar but contains N-acetylglucosamine as the monomer. Both polymers form very similar crystalline macroscopic structures. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones. This is especially true for various fluorescent brightening agents, such as calcofluor white, used as cytochemical markers in microscopic diagnosis of protozoan and fungal infections. A two-domain structural organization is often observed in cellulose-degrading enzymes. Most Trichoderma reesei cellulases consist of a catalytic domain and a cellulose-binding domain (CBD) joined by a linker. The catalytic domain contains the active site with the amino acid residues responsible for the hydrolytic mechanism. The role of the CBD is to bind to the solid cellulose. The ability of CBDs to attach to cellulose can be utilized in various applications. Individual types of CBDs can vary significantly in their properties, such as affinity, preference for crystalline or amorphous cellulose, and cross-reactivity with other similar carbohydrates (7, 8, 9, 10).We have previously described a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of T. reesei cellulase to protozoan cyst wall cellulose (12). In that study we used a recombinant dimeric CBD (D-CBD) fusion protein in an indirect immunofluorescence analysis to specifically stain the cellulose and visualize its localization in the cyst wall. In preliminary studies, this method was also shown to be useful detection of parasites in tissue sections (11).The aim of the present study was to simplify the detection method by preparing D-CBDs as fluorescent and biotinylated conjugates that could be used for direct and rapid detection of cellulose in Acanthamoeba by both fluorescence and ordinary light microscopy.     

Epidemiology of Free-Living Ameba Infections1

Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred world-wide. A majority (144) of these cases have been due to Naegleria fowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (GAE). Acanthamoeba, in addition to causing GAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the human cornea, Acanthamoeba keratitis. Infections due to Acanthamoeba have also been reported in a variety of animals. These observations, together with the fact that Acanthamoeba spp., Naegleria fowleri, and Hartmannella sp. can harbor pathogenic microorganisms such as Legionella and or mycobacteria indicate the public health importance of these amebas.     

Characterization of inhibitors of cellulose synthesis in cotton fibers

Several compounds were tested for their ability to inhibit the in-vivo synthesis of cellulose and other cell-wall polysaccharides in fibers of cotton (Gossypium hirsutum L.) developing on in-vitro cultured ovules. Inhibitory effects were measured by the ability of the compounds to inhibit the incorporation of radioactivity from [U-¹⁴C]glucose into these cell-wall polymers. Of the compounds surveyed, 2,6-dichlorobenzonitrile (DCB) was the most effective and specific one for its effects on cellulose synthesis when compared to its effect on the synthesis of other cell-wall components. At 10 M DCB caused 80% inhibition of cellulose synthesis, and the effect was reversed upon removal of the DCB, with recovery to 90% of the control rate. Two analogs of DCB, 2-chloro-6-fluorobenzonitrile and 2,6-dichlorobenzene carbothiamide, were as specific and nearly as effective as DCB with respect to their effects on cellulose synthesis. Coumarin, generally regarded as an inhibitor of cellulose synthesis in other plant systems, was effective in cotton fibers in millimolar concentrations and, like DCB, was relatively specific with regard to its effect on cellulose synthesis. DCB and coumarin inhibited the synthesis of both primary and secondary wall cellulose. Bacitracin, an inhibitor of the cycling of phosphorylated polyprenols involved in cell-wall synthesis in bacteria, and ethylenediaminetetracetic acid (EDTA) and ethyleneglycol-bis-(-amino-ethylether)-N,N-tetracetic acid (EGTA), chelators of civalent cations, were also effective, although only at relatively high concentrations, in inhibiting incorporation of radioactivity into cellulose.Abbreviations DCB 2,6-dichlorobenzonitrite - CFB 2-chloro-6-fluorobenzonitrile - EDTA ethylenediaminetetracetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)-N,N-tetracetic acid     

Pathogenicity, Morphology, and Differentiation of Acanthamoeba

Acanthamoeba keratitis is sight threatening corneal infection caused by pathogenic Acanthamoeba. Previous studies have shown the genotypic differences between pathogenic and non-pathogenic species/strains of Acanthamoeba. In this study, we examined the morphological differences between pathogenic and non-pathogenic species/strains using scanning electron microscopy. Pathogenic Acanthamoeba exhibited higher number of acanthopodia (structures associated with the binding of amoeba to the target cells) as compared to non-pathogens. In addition, interactions of amoeba with the corneal epithelial cells were studied. Only pathogenic amoeba exhibited adhesion to epithelial cells. Further results indicated that phagocytosis occurs in the pathogenic amoeba by the formation of amoebastome (characteristic of amoeba phagocyte). This study showed that Acanthamoeba phagocytosis may be both an efficient means of obtaining nutrients for the amoeba and a significant factor in the pathogenesis of Acanthamoeba infections. Received: 2 April 2001 / Accepted: 12 April 2001     

Expression levels of encystation mediating factors in fresh strain of Acanthamoeba castellanii cyst ESTs

The life cycle of Acanthamoeba consists of two stages, trophozoite and cyst. The cyst form is resistant to almost all antibiotics. By long term cultivation, Acanthamoeba severely attenuated the encysting ability. To determine the changing of gene expression by the long term cultivation, especially focusing an encystation mediating factors, this study compared the ESTs of the fresh strain and the old strain, and trophozoite. Comparison of the KOG (euKaryotic Orthologous Groups) analysis relative to trophozoite revealed higher percentages of cyst ESTs related to G (Carbohydrate transport and metabolism), H (Coenzyme transport and metabolism), I (Lipid transport and metabolism), D (Cell cycle control, cell division, chromosome partitioning), T (signal transduction mechanisms), and O (Posttranslational modification, protein turnover, chaperones). In addition to this result, KOG analysis of fresh strain relative to old strain showed higher percentage of cyst ESTs related to metabolism category and T (signal transduction mechanisms) article. ESTs of the fresh strain revealed more various gene profiles compared to the old strain including encystation mediating factors like autophagy related proteins (Z article) and signal transduction proteins (T article). Twenty seven kinds of protein kinase C (PKC) like genes were detected in cyst or trophozoite ESTs and twenty one of them were highly expressed during encystation. The information of the expressed genes during encystation in only the fresh strain will provide new clues to understanding the encystation mechanism of encysting protozoa including Acanthamoeba.     

Glycogen phosphorylase in Acanthamoeba spp.: determining the role of the enzyme during the encystment process using RNA interference

Acanthamoeba infections are difficult to treat due to often late diagnosis and the lack of effective and specific therapeutic agents. The most important reason for unsuccessful therapy seems to be the existence of a double-wall cyst stage that is highly resistant to the available treatments, causing reinfections. The major components of the Acanthamoeba cyst wall are acid-resistant proteins and cellulose. The latter has been reported to be the major component of the inner cyst wall. It has been demonstrated previously that glycogen is the main source of free glucose for the synthesis of cellulose in Acanthamoeba, partly as glycogen levels fall during the encystment process. In other lower eukaryotes (e.g., Dictyostelium discoideum), glycogen phosphorylase has been reported to be the main tool used for glycogen breakdown in order to maintain the free glucose levels during the encystment process. Therefore, it was hypothesized that the regulation of the key processes involved in the Acanthamoeba encystment may be similar to the previously reported regulation mechanisms in other lower eukaryotes. The catalytic domain of the glycogen phosphorylase was silenced using RNA interference methods, and the effect of this phenomenon was assessed by light and electron microscopy analyses, calcofluor staining, expression zymogram assays, and Northern and Western blot analyses of both small interfering RNA-treated and control cells. The present report establishes the role of glycogen phosphorylase during the encystment process of Acanthamoeba. Moreover, the obtained results demonstrate that the enzyme is required for cyst wall assembly, mainly for the formation of the cell wall inner layer.     

Acanthamoeba spp.: Efficacy of Bioclen FR One Step®, a povidone-iodine based system for the disinfection of contact lenses

Pathogenic strains of Acanthamoeba are causative agents of a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. The prevalence of this infection has risen in the past 20 years, mainly due to the increase in number of contact lens wearers. Bioclen FR One Step® (Ophtecs Corporation) is the only available povidone-iodine based system for the disinfection of silicone hydrogel lenses and soft contact lenses on the market. Bioclen FR has been proven to be highly effective against bacteria and fungi that can cause problems for contact lens users. In this study, Bioclen FR One Step® was tested against three clinical Acanthamoeba isolates from contact lens cases. The results demonstrated that the tested Acanthamoeba clinical strains were sensitive to Bioclen FR One Step®.     

Use of Recombinant Cellulose-Binding Domains of Trichoderma reesei Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent β-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.     

Increase in XET activity in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) cells habituated to dichlobenil

Bean (Phaseolus vulgaris L.) cells have been habituated to grow in lethal concentrations of dichlobenil (DCB), a specific inhibitor of cellulose biosynthesis. Bean callus cells were successively cultured in increasing DCB concentrations up to 2 μM. The 2-μM DCB habituated cells were impoverished in cellulose and xyloglucan, had an increased xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity, together with an increased growth rate and a decreased molecular size of xyloglucan. However, the application of lethal concentrations of two different cellulose-biosynthesis inhibitors (DCB and isoxaben) for a short period of time produced little effect on XET activity and xyloglucan molecular size. We propose that the weakening of plant cell wall provoked by decrease in cellulose content might promote the xyloglucan tethers and increase the ability of xyloglucan to bind to cellulose in order to give rigidity to the wall.     

The type III secretion system is involved in Escherichia coli K1 interactions with Acanthamoeba

The type III secretion system among Gram-negative bacteria is known to deliver effectors into host cell to interfere with host cellular processes. The type III secretion system in Yersina, Pseudomonas and Enterohemorrhagic Escherichia coli have been well documented to be involved in the bacterial pathogenicity. The existence of type III secretion system has been demonstrated in neuropathogenic E. coli K1 strains. Here, it is observed that the deletion mutant of type III secretion system in E. coli strain EC10 exhibited defects in the invasion and intracellular survival in Acanthamoeba castellanii (a keratitis isolate) compared to its parent strain. Next, it was determined whether type III secretion system plays a role in E. coli K1 survival inside Acanthamoeba during the encystment process. Using encystment assays, our findings revealed that the type III secretion system-deletion mutant exhibited significantly reduced survival inside Acanthamoeba cysts compared with its parent strain, EC10 (P < 0.01). This is the first demonstration that the type III secretion system plays an important role in E. coli interactions with Acanthamoeba. A complete understanding of how amoebae harbor bacterial pathogens will help design strategies against E. coli transmission to the susceptible hosts.     

Down-Regulation of Cellulose Synthase Inhibits the Formation of Endocysts in Acanthamoeba

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.     

Loop-Mediated Isothermal Amplification Targeting 18S Ribosomal DNA for Rapid Detection of Acanthamoeba

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.     

Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.     

Major Role for Cysteine Proteases during the Early Phase of Acanthamoeba castellanii Encystment

Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in A. castellanii is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.The bacteriovorous Acanthamoeba spp. occur ubiquitously in the environment (27) and have a two-stage life cycle consisting of the replicating and feeding trophozoite stage and the dormant, double-walled, cyst stage (16). Cysts are formed in order to survive in an inhospitable environment and are able to persist in a wide variety of habitats (4, 17). Indeed, the ubiquity of Acanthamoeba is made possible by the extreme resistance of the cyst against desiccation, temperature changes, chemicals, radiation, and prolonged starvation. Also, various antiamoebic agents, such as benzalkonium chloride and propamidine isethionate, have no effect on cysts (9, 13, 29). Since acanthamoebae are facultative pathogens that can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE), encystment is also of medical relevance (16). An often occurring complication in the treatment of AK is the presence of viable cysts that remain in the corneal stroma after initial successful therapy, as these can eventually excyst again and lead to recurrent infections (23).According to Weisman (31), the encystment process comprises three phases: induction, wall synthesis, and dormancy. During the induction phase, trophozoites begin to lose their amoeboid appearance and become round. The first wall that is formed gives rise to the exocyst; this wall is 0.3 to 0.5 μm thick and consists mostly of acid-insoluble proteins. The endocyst is formed after the appearance of a well-defined layer whose major component is cellulose (31). Cell wall synthesis is usually accompanied by a decrease in cytoplasmic mass of approximately 80% through a gradual dehydration of the amoeba, thereby causing retraction of the protoplast from the cell wall (2). Rather early, autolysosomes appear and remain in the cytoplasm throughout the whole encystment process. In light of these dramatic changes in the cell''s physiology, it is surprising that the encysting cell can stop and revert the process until 15 h after induction (30). Afterwards, however, cells become committed to the completion of the encystment process.At the molecular level, a number of factors involved in the encystment process have been characterized thus far. For example, cyst-specific protein 21 (Csp21) is a cyst wall protein found in group II acanthamoebae and was reported to be synthesized approximately 12 h after induction (6). The expression of the respective gene is repressed under normal growth conditions via one or more repressor elements between the TATA box and nucleotide (nt) +63 (3). Furthermore, encystment requires serine protease activity (5, 20) and autophagy proteins (22), all of which are suggested to be involved in autolytic processes, and glycogen phosphorylase, which is necessary for the breakdown of glycogen (14). The glucose-1-phosphate that is thereby liberated is subsequently used for the buildup of cellulose in the cyst wall.In the search for additional factors, there have been several successful attempts in the past years to screen encysting Acanthamoeba castellanii for genes specifically expressed during encystment at the mRNA level (19, 21) as well as at the protein level (1, 24). However, there is still a lack of information on the extent of cellular reorganization during the encystment process at the protein level. In this study, we therefore aimed to monitor the encystment process in PAT06, a new clinical isolate of A. castellanii (10), by using two-dimensional gel electrophoresis (2DE) and to analyze the developmental and molecular processes at the proteomic level.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.