Entamoeba Chitinase is Required for Mature Round Cyst Formation.

Entamoeba histolytica, a protozoan parasite, causes amoebiasis in humans. Amoebiasis transmission is solely mediated by chitin-walled cysts, which are produced in the large intestine of humans from proliferative trophozoites by a cell differentiation process called encystation. Resistance to environmental stresses, an essential characteristic for transmission, is attributed to the cyst wall, which is constructed from chitin and several protein components, including chitinase. Chitinase may play a key role in cyst wall formation; however, this has not been confirmed. Here, to elucidate the physiological role of chitinase during Entamoeba encystation, we identified a new chitinase inhibitor, 2,6-dichloro-4-[2-(1-piperazinyl)-4-pyridinyl]-N-(1,3,5-trimethyl-1H-pyrazol-4-yl)-benzenesulfonamide, by recombinant-Entamoeba chitinase-based screening of 400 Pathogen Box chemicals. This compound dose dependently inhibited native chitinase associated with Entamoeba invadens encystation, a model for E. histolytica encystation, with an 50% inhibitory concentration (IC50) of ∼0.6 µM, which is comparable to the IC50s (0.2 to 2.5 µM) for recombinant E. histolytica and E. invadens chitinases. Furthermore, the addition of this compound to E. invadens encystation-inducing cultures increased the generation of cyst walls with an abnormal shape, the most characteristic of which was a "pot-like structure." A similar structure also appeared in standard culture, but at a far lower frequency. These results indicate that chitinase inhibition increases the number of abnormal encysting cells, thereby significantly reducing the efficiency of cyst formation. Transmission electron microscopy showed that compound-treated encysting cells formed an abnormally loose cyst wall and an unusual gap between the cyst wall and cell membrane. Hence, Entamoeba chitinase is required for the formation of mature round cysts. IMPORTANCE Amoebiasis is caused by Entamoeba histolytica infection and is transmitted by dormant Entamoeba cells or cysts. Cysts need to be tolerant to severe environmental stresses faced outside and inside a human host. To confer this resistance, Entamoeba parasites synthesize a wall structure around the cell during cyst formation. This cyst wall consists of chitin and several protein components, including chitinase. The physiological roles of these components are not fully understood. Here, to elucidate the role of chitinase during cyst formation, we identified a new chitinase inhibitor by screening a library of 400 compounds. Using this inhibitor, we showed that chitinase inhibition causes the formation of abnormal cyst walls, the most characteristic of which is a "pot-like structure." This results in decreased production of mature cysts. Chitinase is therefore required for Entamoeba to produce mature cysts for transmission to a new host.

Ultra-structural analysis and morphological changes during the differentiation of trophozoite to cyst in Entamoeba invadens.

Entamoeba histolytica, a pathogenic parasite, is the causative organism of amoebiasis and uses human colon to complete its life cycle. It destroys intestinal tissue leading to invasive disease. Since it does not form cyst in culture medium, a reptilian parasite Entamoeba invadens serves as the model system to study encystation. Detailed investigation on the mechanism of cyst formation, information on ultra-structural changes and cyst wall formation during encystation are still lacking in E. invadens. Here, we used electron microscopy to study the ultrastructural changes during cyst formation and showed that the increase in heterochromatin patches and deformation of nuclear shape were early events in encystation. These changes peaked at ∼20 h post induction, and normal nuclear morphology was restored by 72 h. Two types of cellular structures were visible by 16 h. One was densely stained and consisted of the cytoplasmic mass with clearly visible nucleus. The other consisted of membranous shells with large vacuoles and scant cytoplasm. The former structure developed into the mature cyst while the latter structure was lost after 20 h, This study of ultra-structural changes during encystation in E. invadens opens up the possibilities for further investigation into the mechanisms involved in this novel process.

The dynamics of ultrastructural changes during Entamoeba invadens encystation.

Entamoeba histolytica infection causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of E. histolytica cysts, cysts being the sole form responsible for host-to-host transmission. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed 'encystation'. Therefore, encystation is an important process from a medical as well as a biological perspective. Previous electron microscopy studies have shown the ultrastructure of precysts and mature cysts; however, the dynamics of ultrastructural changes during encystation were ambiguous. Here, we analysed a series of Entamoeba invadens encysting cells by transmission electron microscopy. Entamoeba invadens is a model for encystation and the cells were prepared by short interval time course sampling from in vitro encystation-inducing cultures. We related sampled cells to stage conversion, which was monitored in the overall population by flow cytometry. The present approach revealed the dynamics of ultrastructure changes during E. invadens encystation. Importantly, the results indicate a functional linkage of processes that are crucial in encystation, such as glycogen accumulation and cyst wall formation. Hence, this study provides a reference for studying sequential molecular events during Entamoeba encystation.

Morphological and Motility Features of the Stable Bleb-Driven Monopodial Form of Entamoeba and Its Importance in Encystation.

Entamoeba histolytica and its reptilian counterpart and encystation model Entamoeba invadens formed a polarized monopodial morphology when treated with pentoxifylline. This morphology was propelled by retrograde flow of the cell surface resulting from a cyclic sol-gel conversion of cytoplasm and a stable bleb at the leading edge. Pentoxifylline treatment switched the unpolarized, adherent trophozoites to the nonadherent, stable bleb-driven form and altered the motility pattern from slow and random to fast, directionally persistent, and highly chemotactic. Interestingly, exogenously added adenosine produced multiple protrusions and random motility, an opposite phenotype to that of pentoxifylline. Thus, pentoxifylline, an adenosine antagonist, may be inducing the monopodial morphology by preventing lateral protrusions and restricting the leading edge to one site. The polarized form of E. invadens was aggregation competent, and time-lapse microscopy of encystation revealed its appearance during early hours, mediating the cell aggregation by directional cell migration. The addition of purine nucleotides to in vitro encystation culture prevented the formation of polarized morphology and inhibited the cell aggregation and, thus, the encystation, which further showed the importance of the polarized form in the Entamoeba life cycle. Cell polarity and motility are essential in the pathogenesis of Entamoeba parasites, and the stable bleb-driven polarized morphology of Entamoeba may also be important in invasive amoebiasis.

Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding Proteins.

Early steps of tissue invasion by Entamoeba histolytica are mediated by adhesion and migration through matrix components such as fibronectin with the participation of the actin cytoskeleton. Striking differences in their produced structures, movement, and migration were found. These observations suggest differential changes in their ability to organize the actin cytoskeleton and, therefore, to modify its morphology after adhesion to fibronectin. To understand these observations, we explore deeper the cytoskeleton pathway of E. histolytica compared to Entamoeba dispar, analyzing the activation and involvement of actin cytoskeleton regulatory proteins such as small GTPases (Rho, Rac1 and Cdc42), myosin IB, paxillin, alpha-actinin, and ARP2/3 during interaction with fibronectin. Results showed a higher activation of Rac1 in E. histolytica compared to E. dispar, while Cdc42 and RhoA were equally activated in both amebae; besides, variations in the amount of myosin IB, paxillin, and ARP2/3 were detected among these species, coinciding and reflected in formation of lamellipodia in E. histolytica and filopodia in E. dispar. These could partially explain the higher invasive capacity of E. histolytica compared to E. dispar, due to its pleomorphic ability, high motility, migration, activation, and abundance of proteins involved in the cytoskeleton arrangement.

New advances in scanning microscopy and its application to study parasitic protozoa.

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii, Tritrichomonas foetus, Giardia intestinalis, and Trypanosoma cruzi, compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities.

Development of molecular diagnostic protocols for detecting three types of Entamoeba from diarrheal and asymptomatic pigs and environmental moist soils.

Entamoeba suis and Entamoeba polecki subtypes (ST) 1 and 3 have recently been implicated in disease outbreaks in pigs. However, the distributions of these parasites in Japan and the potential sources of infection on farms still remain unclear. Here, we examined a farm of fattening/growing pigs with abnormal feces in Kagoshima Prefecture, Japan, and found the presence of parasites in the farm environment. Examination of intestinal tissues from pigs presenting with ulcerative colitis revealed a large number of trophozoites that had invaded the lesions. We identified single and mixed infections of E. suis and E. polecki ST1 and ST3 in paraffin sections or fecal samples from affected pigs. Two subtypes of Entamoeba were identified using four primer sets by PCR and sequencing. The parasites were detected in moist soil samples obtained around the drinking water source or puddles, implicating transmission of cysts via contaminated soils. Additionally, we found evidence of Entamoeba spp. and coinfections in surveyed pigs without any diarrhea at two neighboring farms. Our results establish methods for successfully identification of parasites, including cases in which multiple infections are present.

Paleoparasitological analysis of the extinct Myotragus balearicus Bate 1909 (Artiodactyla, Caprinae) from Mallorca (Balearic Islands, Western Mediterranean).

Myotragus balearicus (Artiodactyla, Caprinae) is an extinct caprine endemic of the Eastern Balearic Islands or Gymnesics (i.e., Mallorca, Menorca and surrounding islets, Western Mediterranean Sea). In spite of its small size, c. 50cm height at the shoulder, it was the largest mammal inhabiting these islands until the human arrival, and it had peculiar short legs and frontal vision. It disappeared between 2830 and 2210calBCE. The coprolites here studied were recovered from Cova Estreta, in Pollença, Mallorca. The samples were subjected to microscopic examination and enzyme-linked immunosorbent assays (ELISA) for E. histolytica/E. dispar, Giardia intestinalis and Cryptosporidium parvum. This study provides new paleoparasitological data from an extinct animal species of the Holocene period. The microscopy revealed one sample containing uninucleated-cyst of Entamoeba sp., whereas ELISA detected nine positive samples for Cryptosporidium sp. The finding of these protozoans can help in the discussion of its extinction cause and demonstrates the antiquity and the evolutionary history of host-parasite relationships between protozoa and caprines since the Messinian.

First report of mixed Entamoeba polecki (ST 1) and E. suis infection in piglets shedding abnormalfeces by histopathological and molecular surveys.

Of the three species of Entamoeba found in swine, namely E. histolytica, E. polecki and E. suis, E. histolytica can also infect humans, producing colitis or abscesses of liver and leading to death. However, the pathogenicity of other species of Entamoeba has not been fully characterized. Here, we conducted histopathology and molecular surveys on a pig farm where piglets had blackish feces or muddy diarrhea. Histopathological examination of two piglets showed necrosis of the mucous surface at the ileum, cecum, or colon, infiltration of neutrophils, and formation of ulcers. Based on morphological characteristics, E. polecki and E. suis trophozoites were mainly detected at lamina propria and surface of the lesion, respectively, and Lawsonia intracellularis, a bacterial pathogen, was also detected. Molecular analysis using the small subunit ribosomal RNA gene on other piglets and a sow revealed infection with both E. polecki and E. suis. These findings corroborate our previous reports that the two Entamoeba spp. are pathogenic in pigs as aggravations of symptoms with L. intracellularis. This is the first report about mixed infection with E. polecki and E. suis.

Cell-matrix interactions of Entamoeba histolytica and E. dispar. A comparative study by electron-, atomic force- and confocal microscopy.

Invasion of tissues by Entamoeba histolytica is a multistep process that initiates with the adhesion of the parasite to target tissues. The recognition of the non-invasive Entamoeba dispar as a distinct, but closely related protozoan species raised the question as to whether the lack of its pathogenic potential could be related to a weaker adhesion due to limited cytoskeleton restructuring capacity. We here compared the adhesion process of both amebas to fibronectin through scanning, transmission, atomic force, and confocal microscopy. In addition, electrophoretic and western blot assays of actin were also compared. Adhesion of E. histolytica to fibronectin involves a dramatic reorganization of the actin network that results in a tighter contact to and the subsequent focal degradation of the fibronectin matrix. In contrast, E. dispar showed no regions of focal adhesion, the cytoskeleton was poorly reorganized and there was little fibronectin degradation. In addition, atomic force microscopy using topographic, error signal and phase modes revealed clear-cut differences at the site of contact of both amebas with the substrate. In spite of the morphological and genetic similarities between E. histolytica and E. dispar the present results demonstrate striking differences in their respective cell-to-matrix adhesion processes, which may be of relevance for understanding the invasive character of E. histolytica.

Ultrastructural characteristics and molecular identification of Entamoeba suis isolated from pigs with hemorrhagic colitis: implications for pathogenicity.

Protozoan parasites of the genus Entamoeba infect many classes of vertebrates and are primarily classified based on morphological criteria. To date, only a few species have been proven to cause disease. Here, we examined the pathology of infected pigs with hemorrhage and detected Entamoeba parasites. Isolates were characterized genetically and ultrastructurally to identify the species. Histopathologically, bleeding and thrombus formation were seen only in the large intestine mucosa, where a large number of trophozoites or some Entamoeba cysts were observed around breakdowns in the lamina propria. No screw-shaped bacteria were detected in the lesions, and no pathogenic bacteria such as Brachyspira spp. were detected in fecal cultures. Interestingly, electron microscopy revealed that the parasites possessed mitochondrial organelles, unlike other Entamoeba spp. The isolates were identified as Entamoeba suis by PCR analysis and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. In phylogenetic analyses based on the actin gene, the E. suis isolate formed a cluster with Entamoeba histolytica and Entamoeba invadens, as well as with other parasites of the Amoebidae. Whether the pathogenicity of the E. suis isolate is affected by the severity of infection or host health status remains unclear; however, our results suggest that E. suis could cause or exacerbate clinical symptoms such as hemorrhagic colitis or diarrhea.

Evolution of Archamoebae: morphological and molecular evidence for pelobionts including Rhizomastix, Entamoeba, Iodamoeba, and Endolimax.

The archamoebae form a small clade of anaerobic/microaerophilic flagellates or amoebae, comprising the pelobionts (mastigamoebids and pelomyxids) and the entamoebae. It is a member of the eukaryotic supergroup Amoebozoa. We examined 22 strains of 13 species of Mastigamoeba, Pelomyxa and Rhizomastix by light-microscopy and determined their SSU rRNA gene sequences. The SSU rRNA gene sequences of Pelomyxa palustris and Mastigella commutans in GenBank are shown to belong to P. stagnalis and Mastigamoeba punctachora, respectively. Five new species of free-living archamoebae are described: Mastigamoeba abducta, M. errans, M. guttula, M. lenta, and Rhizomastix libera spp. nov. A species of Mastigamoeba possibly living endosymbiotically in Pelomyxa was identified. Rhizomastix libera, the first known free-living member of that genus, is shown to be an archamoeba. R. libera possesses an ultrastructure unique within archamoebae: a rhizostyle formed from a modified microtubular cone and a flagellum with vanes. While many nominal species of pelobionts are extremely hard to distinguish by light microscopy, transient pseudopodial characters are worthy of further investigation as taxonomic markers.

Vitrification of thick samples for soft X-ray cryo-tomography by high pressure freezing.

Soft X-ray cryo-microscopy (cryo-XT) offers an ideal complement to electron cryo-microscopy (cryo-EM). Cryo-XT is applicable to samples more than an order of magnitude thicker than cryo-EM, albeit at a more modest resolution of tens of nanometers. Furthermore, the natural contrast obtained in the "water-window" by differential absorption by organic matter vs water yields detailed images of organelles, membranes, protein complexes, and other cellular components. Cryo-XT is thus ideally suited for tomography of eukaryotic cells. The increase in sample thickness places more stringent demands on sample preparation, however. The standard method for cryo-EM, i.e., plunging to a cryogenic fluid such as liquid ethane, is no longer ideally suited to obtain vitrification of thick samples for cryo-XT. High pressure freezing is an alternative approach, most closely associated with freeze-substitution and embedding, or with electron cryo-microscopy of vitreous sections (CEMOVIS). We show here that high pressure freezing can be adapted to soft X-ray tomography of whole vitrified samples, yielding a highly reliable method that avoids crystallization artifacts and potentially offers improved imaging conditions in samples not amenable to plunge-freezing.

Mitosomes in trophozoites and cysts of the reptilian parasite Entamoeba invadens.

Heat shock protein genes led to the discovery of mitosomes in Entamoeba histolytica, but mitosomes have not been described for any other Entamoeba species, nor have they been identified in the cyst stage. Here, we show that the distantly related reptilian pathogen Entamoeba invadens contains mitosomes, in both trophozoites and cysts, suggesting all Entamoeba species contain these organelles.

High-resolution electron microscopical study of cyst walls of Entamoeba spp.

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.

The nucleolus in Entamoeba histolytica and Entamoeba invadens is located at the nuclear periphery.

The ribosomal RNA genes in the human parasite Entamoeba histolytica and its reptilian counterpart Entamoeba invadens are located on extrachromosomal circles. The expression of rRNA genes generally takes place in a specialized nuclear compartment-the nucleolus. In Entamoeba species the nuclear space that may be called the nucleolus has yet to be defined. Previous studies showed that the rDNA circles are located at the nuclear periphery. Here we have raised antibodies against the E. histolytica homologue of fibrillarin, a highly conserved protein known to be a marker for nucleolus. These antibodies cross-reacted preferentially with the nuclear periphery, forming a peripheral ring. There was complete colocalization of fibrillarin with the signal obtained by antibodies against E. histolytica RNA polymerase I (but not polymerase II and III), strongly suggesting that the nucleolus in E. histolytica is indeed located at the nuclear periphery. The dynamic nature of the nucleolus was evident when cells were subjected to a variety of growth stresses. Although the peripheral nucleolar structure was retained, stress was accompanied by significant cytoplasmic localization of RNA polymerase I, and to some extent fibrillarin. The nucleolus in E. invadens was also located at the nuclear periphery. When these cells were induced to encyst the nucleolar ring structure was lost, giving way to small, fragmented foci. This study gives the first clear insight into nucleolar structure in Entamoeba.

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa.

Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania.

Strategies and results of field emission scanning electron microscopy (FE-SEM) in the study of parasitic protozoa.

Field emission scanning electron microscopy (FE-SEM) provides a range of strategies for investigating the structural organization of biological systems, varying from isolated macromolecules to tissue organization and whole organisms. This review covers some of the results so far obtained using FE-SEM observation and various protocols of sample fixation to analyze the structural organization of parasitic protozoa and their interaction with host cells. The employment of FE-SEM can be broadened through the use of gold-labeled molecules or tracers, gradual extraction by detergents, and cleavage techniques. These analyses provide significant contributions to the characterization of these organisms concerning ultrastructure, cytoskeleton, motility and intracellular behavior.

Entamoeba invadens: restriction of ploidy by colonic short chain fatty acids.

The DNA content of Entamoeba parasites appears to be regulated by an unusual mechanism. This conclusion, however, was based on experiments that examined parasites grown in media that did not contain short chain fatty acids (SCFAs) normally found in the colonic lumen. Since one of these SCFAs, butyrate, is known to affect DNA replication in eukaryotic cells, we examined the effect of SCFAs on Entamoeba trophozoite DNA content. Similar to reports from others, we found that Entamoeba invadens trophozoite cultures grown in conventional medium (TYI-S-33) contained cells with 2N, 4N, 8N, and 16N amounts of DNA. In contrast, cultures grown in TYI medium containing colonic SCFAs added in place of glucose contained a minor population with 2N, a major population with 4N, and very few cells with higher amounts of DNA. SCFAs also prevented the normal increase in the number of nuclei per cell in trophozoites that were induced to encyst. These results suggest that E. invadens trophozoite stage parasites growing in the intestine in the presence of high amounts of SCFAs have a ploidy range restricted to 2N/4N. Axenic growth of trophozoites in the absence of SCFAs, however, appears to allow trophozoites to increase the amount of DNA per cell, which they must do during the normal encystment process.