Ceramide synthase 2 knockdown suppresses trophozoite growth, migration, in vitro encystment and excystment of Entamoeba invadens.

Entamoeba invadens is the protozoan which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment/excystment in vitro. Here we report that EinCerS2 knockdown promoted decrease in sphingomyelin (SM) subspecies with long-chain fatty acids (24:0) down to 50% but increase sphingolipids with short-chain fatty acids (16:0) up to three times in both trophozoites and cysts of E. invadens. EinCerS2 silencing also resulted in decreased trophozoites' movement, proliferation, cysts formation, and trophozoites hatched after excystment. By immunofluorescence assays, a polyclonal antibody against EinCerS2 detected the enzyme in the cytoplasm of E. invadens trophozoites, colocalizing with Endoplasmic Reticulum-resident cognate EiSERCA. Interestingly, EinCerS2 was redistributed close to the plasma membrane during encystation, suggesting that the generation of diacylglycerol (DAG) via synthesis of sphingolipids and the activation protein kinase C might participate in the encystment process of E. invadens.

Entamoeba marina n. sp.; a New Species of Entamoeba Isolated from Tidal Flat Sediment of Iriomote Island, Okinawa, Japan.

The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediment collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 µm in length and 6.8-15.9 µm in width, whereas the cysts were 8.9-15.8 µm in diam. and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection sites. Therefore, we propose E. marina as a new species of the genus Entamoeba.

Identification of Entamoeba polecki with Unique 18S rRNA Gene Sequences from Celebes Crested Macaques and Pigs in Tangkoko Nature Reserve, North Sulawesi, Indonesia.

Unique species of macaques are distributed across Sulawesi Island, Indonesia, and the details of Entamoeba infections in these macaques are unknown. A total of 77 stool samples from Celebes crested macaques (Macaca nigra) and 14 stool samples from pigs were collected in Tangkoko Nature Reserve, North Sulawesi, and the prevalence of Entamoeba infection was examined by PCR. Entamoeba polecki was detected in 97% of the macaques and all of the pigs, but no other Entamoeba species were found. The nucleotide sequence of the 18S rRNA gene in E. polecki from M. nigra was unique and showed highest similarity with E. polecki subtype (ST) 4. This is the first case of identification of E. polecki ST4 from wild nonhuman primates. The sequence of the 18S rRNA gene in E. polecki from pigs was also unique and showed highest similarity with E. polecki ST1. These results suggest that the diversity of the 18S rRNA gene in E. polecki is associated with differences in host species and geographic localization, and that there has been no transmission of E. polecki between macaques and pigs in the study area.

Evidence for a "wattle and daub" model of the cyst wall of entamoeba.

The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a "wattle and daub" model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).

Entamoeba invadens: dynamics of DNA synthesis during differentiation from trophozoite to cyst.

The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba invadens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the number of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0-8h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commitment phase (8-24h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucleated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24-72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be getting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucleated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens.

Epidemiology, species composition and genetic diversity of tetra- and octonucleated Entamoeba spp. in different Brazilian biomes.

BACKGROUND: Entamoeba species harbored by humans have different degrees of pathogenicity. The present study explores the intra- and interspecific diversity, phylogenetic relationships, prevalence and distribution of tetra- and octonucleated cyst-producing Entamoeba in different Brazilian regions. METHODS: Cross-sectional studies were performed to collect fecal samples (n = 1728) and sociodemographic data in communities located in four Brazilian biomes: Atlantic Forest, Caatinga, Cerrado, and Amazon. Fecal samples were subjected to molecular analysis by partial small subunit ribosomal DNA sequencing (SSU rDNA) and phylogenetic analysis. RESULTS: Light microscopy analysis revealed that tetranucleated cysts were found in all the studied biomes. The highest positivity rates were observed in the age group 6-10 years (23.21%). For octonucleated cysts, positivity rates ranged from 1 to 55.1%. Sixty SSU rDNA Entamoeba sequences were obtained, and four different species were identified: the octonucleated E. coli, and the tetranucleated E. histolytica, E. dispar, and E. hartmanni. Novel haplotypes (n = 32) were characterized; however, new ribosomal lineages were not identified. The Entamoeba coli ST1 subtype predominated in Atlantic Forest and Caatinga, and the ST2 subtype was predominant in the Amazon biome. E. histolytica was detected only in the Amazon biome. In phylogenetic trees, sequences were grouped in two groups, the first containing uni- and tetranucleated and the second containing uni- and octonucleated cyst-producing Entamoeba species. Molecular diversity indexes revealed a high interspecific diversity for tetra- and octonucleated Entamoeba spp. (H ± SD = 0.9625 ± 0.0126). The intraspecific diversity varied according to species or subtype: E. dispar and E. histolytica showed lower diversity than E. coli subtypes ST1 and ST2 and E. hartmanni. CONCLUSIONS: Tetra- and octonucleated cyst-producing Entamoeba are endemic in the studied communities; E. histolytica was found in a low proportion and only in the Amazon biome. With regard to E. coli, subtype ST2 was predominant in the Amazon biome. The molecular epidemiology of Entamoeba spp. is a field to be further explored and provides information with important implications for public health.

Sub-pixel resolving optofluidic microscope for on-chip cell imaging.

We report the implementation of a fully on-chip, lensless, sub-pixel resolving optofluidic microscope (SROFM). The device utilizes microfluidic flow to deliver specimens directly across a complementary metal oxide semiconductor (CMOS) sensor to generate a sequence of low-resolution (LR) projection images, where resolution is limited by the sensor's pixel size. This image sequence is then processed with a pixel super-resolution algorithm to reconstruct a single high resolution (HR) image, where features beyond the Nyquist rate of the LR images are resolved. We demonstrate the device's capabilities by imaging microspheres, protist Euglena gracilis, and Entamoeba invadens cysts with sub-cellular resolution and establish that our prototype has a resolution limit of 0.75 microns. Furthermore, we also apply the same pixel super-resolution algorithm to reconstruct HR videos in which the dynamic interaction between the fluid and the sample, including the in-plane and out-of-plane rotation of the sample within the flow, can be monitored in high resolution. We believe that the powerful combination of both the pixel super-resolution and optofluidic microscopy techniques within our SROFM is a significant step forwards toward a simple, cost-effective, high throughput and highly compact imaging solution for biomedical and bioscience needs.

Differential identification of Entamoeba spp. based on the analysis of 18S rRNA.

Entamoeba histolytica is known to cause intestinal and extra-intestinal disease while the other Entamoeba species are not considered to be pathogenic. However, all Entamoeba spp. should be reported when identified in clinical samples. Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features overlap. E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii are morphologically identical but can be differentiated using molecular tools. We developed a polymerase chain reaction (PCR) procedure followed by DNA sequencing of specific regions of 18S rRNA gene to differentiate the Entamoeba spp. commonly found in human stools. This approach was used to analyze 45 samples from cases evaluated for the presence of Entamoeba spp. by microscopy and a real-time PCR method capable of differential detection of E. histolytica and E. dispar. Our results demonstrated an agreement of approximately 98% (45/44) between the real-time PCR for E. histolytica and E. dispar and the 18S rRNA analysis described here. Five previously negative samples by microscopy revealed the presence of E. dispar, E. hartmanii, or E. coli DNA. In addition, we were able to detect E. hartmanii in a stool sample that had been previously reported as negative for Entamoeba spp. by microscopy. Further microscopic evaluation of this sample revealed the presence of E. hartmanii cysts, which went undetected during the first microscopic evaluation. This PCR followed by DNA sequencing will be useful to refine the diagnostic detection of Entamoeba spp. in stool and other clinical specimens.

'Entamoeba histolytica' identified by stool microscopy from children with acute diarrhoea in Peru is not E. histolytica.

Entamoeba histolytica is a rare but feared pathogen owing to its related morbidity and mortality. Physicians in an ambulatory clinic in Cusco noted frequent reports of E. histolytica diagnosed by microscopy. Other non-pathogenic species of Entamoeba have an identical microscopic appearance. To determine whether the organisms were actually E. histolytica, faecal specimens from children aged six months to three years with diarrhoea were tested by a species-specific ELISA for E. histolytica antigen. Although 19/73 patients (26.0%) were presumptively diagnosed with amoebiasis based on microscopy, none were confirmed by ELISA. Most cases diagnosed as E. histolytic by microscopy in Peru are not infected by the pathogenic species and are probably colonised by non-pathogenic amoeba such as Entamoeba dispar.

Structure of ribosomes of chromatoid bodies: three-dimensional Fourier synthesis at low resolution.

A three-dimensional Fourier synthesis at an axial resolution of 75 angstroms of optical diffraction data from electron micrographs of stained sections of chromatoid bodies reveals the position of the large and small ribosomal subunits within these crystals of ribosomes.

Entamoeba invadens: sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation.

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous D-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.

Autophagy during proliferation and encystation in the protozoan parasite Entamoeba invadens.

Autophagy is one of the three systems responsible for the degradation of cytosolic proteins and organelles. Autophagy has been implicated in the stress response to starvation, antigen cross-presentation, the defense against invading bacteria and viruses, differentiation, and development. Saccharomyces cerevisiae Atg8 and its mammalian ortholog, LC3, play an essential role in autophagy. The intestinal protozoan parasite Entamoeba histolytica and a related reptilian species, Entamoeba invadens, possess the Atg8 conjugation system, consisting of Atg8, Atg4, Atg3, and Atg7, but lack the Atg5-to-Atg12 conjugation system. Immunofluorescence imaging revealed that polymorphic Atg8-associated structures emerged in the logarithmic growth phase and decreased in the stationary phase and also increased in the early phase of encystation in E. invadens. Immunoblot analysis showed that the increase in phosphatidylethanolamine-conjugated membrane-associated Atg8 was also accompanied by the emergence of Atg8-associated structures during the proliferation and differentiation mentioned above. Specific inhibitors of class I and III phosphatidylinositol 3-kinases simultaneously inhibited both the growth of trophozoites and autophagy and also both encystation and autophagy in E. invadens. These results suggest that the core machinery for autophagy is conserved and plays an important role during proliferation and differentiation in Entamoeba.

Novel Entamoeba Findings in Nonhuman Primates.

In addition to well-known human-infecting species, Entamoeba species not found in humans have been identified recently in nonhuman primates (NHPs). Importantly, it has become clear that the organism identified as Entamoeba histolytica in NHPs is usually a distinct species, Entamoeba nuttalli. Many DNA-based stool surveys use species-specific detection methods and so may miss the full range of Entamoeba species present. In addition, authors may be using the same species name to describe distinct organisms. These various shortcomings may not be obvious to readers. In this review, we clarify the relationships between Entamoeba species' names based on morphological and molecular data, and highlight gaps in recently published data on Entamoeba species in wild NHPs resulting from the use of variable methodology.

Molecular diagnosis of Entamoeba spp. versus microscopy in the Great Cairo.

Amoebiasis is a human disease produced by Entamoeba histolytica which causes widespread mortality and morbidity worldwide through diarrheal disease and abscess establishment in parenchymal tissues such as liver, lung, and brain. The true prevalence of infection is unknown for most areas of the world due to the difficulty to characterise Entamoeba histolytica versus other non-pathogenic amoebas with identical morphology, as Entamoeba dispar, and Entamoeba moshkovskii. To overcome microscopy misidentification issues, we tested a nested multiplex polymerase chain reaction (PCR) and a real-time PCR on 194 stool samples collected from incoming dysentery patients in Cairo hospitals diagnosed with E. histolytica by microscopy. Nested PCR showed only 20 (10.3%) samples positive to E. histolytica and 17 (8.7%) to E. dispar. The real-time PCR detected only 19 and 11 samples positive to E. histolytica and E. dispar respectively, showing less sensitivity than the nested PCR. The data show that prevalence of E. histolytica in Cairo is lower when specific diagnosis methods are used instead of traditional microscopy, allowing to differentiate between morphologically identical human amoebas species.

Morphological and molecular characterization of an uninucleated cyst-producing Entamoeba spp. in captured Rangeland goats in Western Australia.

Uninucleated Entamoeba cysts measuring 7.3×7.7µm were detected in faecal samples collected from wild Rangeland goats (Capra hircus) after arrival at a commercial goat depot near Geraldton, Western Australia at a prevalence of 6.4% (8/125). Sequences were obtained at the 18S rRNA (n=8) and actin (n=5) loci following PCR amplification. At the 18S locus, phylogenetic analysis grouped the isolates closest with an E. bovis isolate (FN666250) from a sheep from Sweden with 99% similarity. At the actin locus, no E. bovis sequences were available, and the isolates shared 94.0% genetic similarity with E. suis from a pig in Western Japan. This is the first report to describe the morphology and molecular characterisation of Entamoeba from Rangeland goats in Western Australia and the first study to produce actin sequences from E. bovis-like Entamoeba sp.

Overdiagnosis of Entamoeba histolytica and Entamoeba dispar in Nicaragua: a microscopic, triage parasite panel and PCR study.

BACKGROUND: Based on stool microscopy, an E. histolytica/E. dispar prevalence of 18.6% was found in León, Nicaragua about 10 years ago. Since then, new non-microscopic methods have been developed to discriminate between pathogenic E. histolytica and nonpathogenic E. dispar. The main objectives of the present study were to evaluate the true prevalence of E. histolytica among individuals with diarrhea and to assess the diagnostic procedures carried out at the health center level. METHODS: A descriptive study was carried out on patients with diarrhea. Parasite detection was performed by conventional microscopy on native preparations or concentrated and stained specimens, Triage Parasite Panel and by PCR for both E. histolytica and E. dispar. RESULTS: In 134 individuals with diarrhea, the prevalence of intestinal parasites was 69% as detected by direct stool examination. E. histolytica/E. dispar was found in eight (6%) of the samples, but the health centers reported 24%. In the Triage Parasite Panel only one case of E. histolytica/E. dispar was found. Analysis by PCR showed E. dispar in ten (7.5%) and E. histolytica in two cases (1.5%). The detection of intestinal coccidia and Dientamoeba fragilis required additional staining methods. CONCLUSIONS: PCR results showed that E. histolytica is a rare finding in patients with diarrhea. At the health centers, E. histolytica, E. histolytica/E. dispar were clearly overdiagnosed, with the consequence of overtreatment.

The cell cycle of Entamoeba invadens during vegetative growth and differentiation.

The cell division cycle of Entamoeba invadens was studied during vegetative growth of trophozoites and during their differentiation into cysts. During vegetative growth of trophozoites, it was observed that DNA synthesis typically continued after one genome content had been duplicated. During encystation, DNA synthesis was arrested after 4n genome content had been synthesised. Using multi-parameter flow cytometry, the light scattering properties of cysts and trophozoites were studied. The cytoplasmic granularity, reflected by the side scatter of light, was proportional to DNA content of trophozoites, whereas cysts with similar DNA contents showed heterogeneity in their cytoplasmic granularity. Dynamic changes in the intracellular calcium pools were observed during differentiation of trophozoites to cysts. Comparison of E. invadens and Entamoeba histolytica cell cycles suggest that both organisms may have similar regulatory processes during cell division and differentiation. Since E. histolytica cannot be induced to encyst in axenic culture, analysis of the E. invadens cell cycle during encystation may be useful for identifying homologous processes in E.histolytica.

Frequency of Entamoeba gingivalis in human gingival scrapings.

A survey was made of gingival scrapings stained by the Papanicolaou method to assess the occurrence of Entamoeba gingivalis, a nonpathogenic-oral amoeba. Positive findings were recorded in 59% of 113 dental patients, and 32% of 96 healthy controls. These figures showed no significant changes during the last 20 years when compared with data published in 1960 and 1963. The existence of E. gingivalis and its rare appearance in the sputum should be known to cytologists because of the morphologic resemblance to Entamoeba histolytica, a pathogenic amoeba. Morphologic features are described to differentiate E. gingivalis from similar structures found in sputum.

Laboratory diagnosis of amebiasis.

The procedure used to diagnose amebiasis varies depending on the geographic location of the laboratory. However, in general, the microscopic diagnosis and differentiation of E. histolytica from other intestinal amebae is most satisfactorily achieved when the specimen is preserved immediately and both a concentration and stained smear are examined. The number of additional positive findings achieved by the cultivation of fecal material does not justify the time and cost involved. On the other hand, serology is a useful adjunct to diagnosis, especially in patients with extraintestinal amebiasis. The commercial development of antigen detection kits and DNA probes may provide more rapid, accurate, and less costly diagnostic procedures for the future. New guidelines need to be formulated regarding the number of specimens to be submitted, and the cost effectiveness of various diagnostic procedures should be evaluated.