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Developing countries continuously face challenges to get rid of amoebiasis, a protozoan disease caused by Entamoeba histolytica. Every year around 900 million people get affected by amoebiasis, among them only 10 % of people show the symptoms of the disease while 90 % of people do not show any symptoms but still, serve as carriers of the disease. Asymptomatic persons carry cysts of Entamoeba in their fecal matter, which is carried by house flies to contaminate the food and water. Entamoeba histolytica is a very successful pathogen because it has very well-developed virulence factors that function in infection to host as well as in overcoming the host's immune response. However, researchers have very little information about the clear relationship between virulence factors and the virulence of Entamoeba histolytica , through various research, researchers have been able to identify key pathogenic factors that are crucial to the pathogenesis of amoebiasis and have provided valuable insights into the development of the disease. The objective of this review is to underscore various virulence factors (Monosaccharides, Gal/GalNAc lectin, extracellular vesicles, cysteine proteases, amoeba-pores, and actin microfilament) involved in pathogenesis which may be helpful for designing of future drug or therapy. [Display omitted] • E. histolytica, successful pathogen because of well-developed virulence factors. • Virulence factors includes Monosaccharide, Gal/GalNAc lectin, EVs, cysteine proteases, amoeba-pore, and actin microfilament. • Gal/GalNAc lectin interaction mediate the attachment of trophozoite to host cell. • E. histolytica uses a unique mechanism called trogocytosis to kill the host cell. [ABSTRACT FROM AUTHOR]

Amoebiasis is a disease caused by Entamoeba histolytica , affecting the large intestine of humans and occasionally leading to extra-intestinal lesions. Entamoeba dispar is another amoeba species considered commensal, although it has been identified in patients presenting with dysenteric and nondysenteric colitis, as well as amoebic liver abscess. Amoebic virulence factors are essential for the invasion and development of lesions. There is evidence showing that the association of enterobacteria with trophozoites contributes to increased gene expression of amoebic virulence factors. Enteropathogenic Escherichia coli is an important bacterium causing diarrhea, with high incidence rates in the world population, allowing it to interact with Entamoeba sp. in the same host. In this context, this study aims to evaluate the influence of enteropathogenic Escherichia coli on ACFN and ADO Entamoeba dispar strains by quantifying the gene expression of virulence factors, including galactose/N-acetyl-D-galactosamine-binding lectin, cysteine proteinase 2, and amoebapores A and C. Additionally, the study assesses the progression and morphological aspect of amoebic liver abscess and the profile of inflammatory cells. Our results demonstrated that the interaction between EPEC and ACFN Entamoeba dispar strains was able to increase the gene expression of virulence factors, as well as the lesion area and the activity of the inflammatory infiltrate. However, the association with the ADO strain did not influence the gene expression of virulence factors. Together, our findings indicate that the interaction between EPEC, ACFN, and ADO Entamoeba dispar strains resulted in differences in vitro and in vivo gene expression of Gal/GalNAc-binding lectin and CP2, in enzymatic activities of MPO, NAG, and EPO, and consequently, in the ability to cause lesions. [Display omitted] • E. dispar strains respond differently to association with EPEC. • E. dispar strains are capable of causing liver damage. • The interaction between EPEC and E. dispar strains elicited variations in amoebic virulence and pathogenicity. [ABSTRACT FROM AUTHOR]

Abstract Entamoeba histolytica harbors an extensive intracellular distribution of ubiquitin-proteasome systems important for numerous cellular processes. However, glycosylation studies of ubiquitin-proteasome components have not yet been elucidated. Here we report the partial characterization of N- linked glycosylation profile in E. histolytica ubiquitin by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), Nanoelectrospray Ionization-Tandem Mass Spectrometry (NSI-MS), Matrix-Assisted Laser-Desorption time-of-flight Mass Spectrometry (MALDI-TOF MS) and Gas Chromatography–Mass Spectrometry (GC–MS) analysis. To our knowledge, the data presented in this report represents the first structural glycomics analysis of E. histolytica ubiquitin, while most of the reports are performed on whole parasitic glycan profiles. The glycan profile of E. histolytica ubiquitin has high mannose N- glycan structures. The N -linked glycan profile showed fragments from Hex 3 HexNAc 2 to Hex 9 HexNAc 2. Based in our findings and ubiquitin function, we hypothesize that the same ubiquitin Asn-Asp-Ser sequon carries heterogenic glycosylations, at different metabolic pathway stages according to ubiquitin functional requirements. Finally, we propose a set of possible high mannose N- glycan structures that will help to elucidate the ubiquitin biochemical composition and may well represent good targets for anti-amoebic drugs. Graphical abstract Image Highlights • The glycan profile of E. histolytica ubiquitin have high mannose N- glycan structures. • The N -linked glycan profile showed fragments from Hex 3 HexNAc 2 to Hex 9 HexNAc 2. • The E. histolytica ubiquitin was preserved from degradation without protease inhibitors. • This is the first report of glycans in E. histolytica ubiquitin. Most studies are performed on whole parasitic glycan. [ABSTRACT FROM AUTHOR]

Abstract Amoebiasis is a worldwide health problem caused by the pathogen Entamoeba histolytica. Several virulence factors have been implicated in host invasion, immune evasion, and tissue damage. There are still new factors that remain to be elucidated and characterized. In this work, we obtained amoebic transfectants overexpressing three of the neutral sphingomyelinase enzymes encoded in the E. histolytica genome. The EhnSM3 overexpression induced an increase in hemolytic and cytotoxic activities, besides an increase in gene expression of amoebapore A, B, and C. Meanwhile the EhnSM1 and EhnSM2 overexpression caused an increase in cytopathic activity. In all the neutral sphingomyelinases overexpressing strains, the gene expression levels for cysteine proteinase 5, adhesin 112 and, heavy and light Gal/GalNAc lectin subunits were not affected. We propose that the increase of cytotoxic and lytic effect of EhnSM3 overexpressed strain can be related to the sum of the effect of EhnSM3 plus amoebapores, in a process cell contact-dependent or as mediator by inducing the gene expression of amoebapores enabling a link between EhnSM3 with the virulence phenotype in E. histolytica. Our results suggest a differential role for neutral sphingomyelinases in E. histolytica virulence. Graphical abstract Image 1 Highlights • Amoebic transfectants overexpress the three neutral sphingomyelinase enzymes encoded in the E. histolytica genome. • The EhnSM1 and EhnSM2 overexpression in trophozoites is related with an increase of monolayer destruction of target cells. • The EhnSM3 overexpression induced an increase in hemolytic and cytotoxic activities of E. histolytica. • The EhnSM3 overexpression in E. histolytica is related with an increase of gene expression of amoebapores. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica genetic organization and genome structure is complex and under intense research. The genome is fully sequenced, and several tools have been developed for the molecular study of this organism. Nevertheless, good protein tracking tags that are easy to measure and image, like the fluorescent proteins are lacking. In this report, we codon-optimized the red fluorescent protein from the coral Discosoma striata (DsRFP) for its use in E. histolytica and demonstrated functionality in vivo . We envision that this protein can be widely used for the development of transcriptional reporter systems and protein-tagging applications. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica is an intestinal parasite causing significant morbidity and mortality in the developing world. More tools are needed to understand the epidemiology and molecular pathogenesis of amebiasis. Virulence pattern of E. histolytica could be linked with the genotype of a strain. Several loci showing insertion polymorphism of retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2 have been reported among laboratory strains of E. histolytica. The present study was undertaken to validate this observation in clinical isolates from north India. Our results indicate that the Indian samples show a different propensity of SINE retention or loss at two of the polymorphic loci compared with non-Indian samples. Statistical analysis of different loci revealed Locus 17 of EhSINE1as a potential geographical marker for distinguishing Indian isolates from non Indian isolates. [ABSTRACT FROM AUTHOR]

Mucosal-associated parasites, such as Giardia intestinalis , Entamoeba histolytica, and Trichomonas vaginalis , have significant clinical relevance. The pathologies associated with infection by these parasites are among those with the highest incidence of gastroenteritis (giardiasis and amoebiasis) and sexually transmitted infections (trichomoniasis). The treatment of these diseases is based on drugs that act on the anaerobic metabolism of these parasites, such as nitroimidazole and benzimidazole derivatives. One interesting feature of parasites is their ability to produce ATP under anaerobic conditions. Due to the absence of enzymes capable of producing ATP under anaerobic conditions in the vertebrate host, they have become interesting therapeutic targets. This review discusses anaerobic energy metabolism in mucosal-associated parasites, focusing on the anaerobic metabolism of pyruvate, the importance of these enzymes as therapeutic targets, and the importance of treating their infections. [Display omitted] • Hydrogenosome is a specific organelle responsible for the anaerobic ATP synthesis. • Pyruvate ferredoxin oxidoreductase catalyzes the decarboxylation of pyruvate. • Pyruvate ferredoxin oxidoreductase activity is essential to activate nitroimidazoles. [ABSTRACT FROM AUTHOR]

The parasympathetic nervous system has a crucial role in immunomodulation of the vagus nerve, its structure provides a pathogen detection system, and a negative feedback to the immune system after the pathogenic agent has been eliminated. Amebiasis is a disease caused by the protozoan parasite Entamoeba histolytica , considered the third leading cause of death in the world. The rats are used as a natural resistance model to amoebic liver infection. The aim of this study is to analyze the interaction of Entamoeba histolytica with neutrophils, macrophages, and NK cells in livers of intact and vagotomized rats. Six groups were studied (n = 4): Intact (I), Intact + amoeba (IA), Sham (S), Sham + amoeba (SA), Vagotomized (V) and Vagotomized + amoeba (VA). Animals were sacrificed at 8 h post-inoculation of E. histolytica . Then, livers were obtained and fixed in 4% paraformaldehyde. Tissue liver slides were stained with H-E, PAS and Masson. The best development time for E. histolytica infection was at 8 h. Amoeba was identified with a monoclonal anti-220 kDa E. histolytica lectin. Neutrophils (N) were identified with rabbit anti-human neutrophil myeloperoxidase, macrophages (Mɸ) with anti-CD68 antibody and NK cells (NK) with anti-NK. Stomachs weight and liver glycogen were higher in V. Collagen increased in VA, whereas vascular and neutrophilic areas were decreased. There were fewer N, Mɸ, NK around the amoeba in the following order IA > SA > VA (p < 0.05 between IA and VA). In conclusion, these results suggest that the absence of parasympathetic innervation affects the participation of neutrophils, macrophages and NK cells in the innate immune response, apparently by parasympathetic inhibition on the cellular functions and probably for participation in sympathetic activity. [ABSTRACT FROM AUTHOR]

It is known that the microtubules (MT) of Entamoeba histolytica trophozoites form an intranuclear mitotic spindle. However, electron microscopy studies and the employment of anti-beta-tubulin (β-tubulin) antibodies have not exhibited these cytoskeletal structures in the cytoplasm of these parasites. The purpose of this work was to detect β-tubulin in the cytoplasm of interphasic E . histolytica trophozoites. Activated or non-activated HMI-IMSS-strain E . histolytica trophozoites were used and cultured for 72 h at 37 °C in TYI-S-33 medium, and then these were incubated with the anti-β-tubulin antibody of E . histolytica . The anti-β-tubulin antibody reacted with the intranuclear mitotic spindle of E . histolytica- activated trophozoites as control. In contrast, in non-activated interphasic parasites, anti-β-tubulin antibody reacted with diverse puntiform structures in the cytoplasm and with ring-shaped structures localized in the cytoplasm, cellular membrane and endocytic stomas. In this work, for the first time, the presence of β-tubulin is shown in the cytoplasm of E . histolytica trophozoites. [ABSTRACT FROM AUTHOR]

Amoebiasis is the third cause of death due to parasites in the world. Although, numerous serodiagnostic and salivary tests have been developed, the majority of these assays lack sensitivity in endemic zones to detect acute amoebic liver abscess. The two main limiting factors to develop reliable assays are the high levels of anti-amoeba antibodies in populations living in endemic zones, and the proteolysis of amoebic extracts even treated with inhibitors. Our group reported a method to preserve amoebic antigens without using enzymatic inhibitors (IC:MC fraction) that shows stability for years. Here we describe the development of a serologic ELISA to diagnose amoebiasis made with IC: MC antigens, and its validation for clinical use in endemic areas. In our study, we included sera from 66 patients diagnosed with acute amoebic liver abscess and 33 volunteers living in an endemic area for amoebiasis. Our assay was compared with an indirect haemaglutination assay (IHA) an ELISA elaborated with antigens derived from untreated trophozoites. The ELISA made with IC: MC antigens presented more reproducibility compared to other assays. Sera from 95% ALA patients showed a positive value. The ELISA (IC: MC) detected 97% of patients with ALA compared to an 81% using IHA. The parameters of ELISA (vs. IHA) were Sensitivity 98% (81%), Specificity 96% (97%), Positive predictive value 98% (96%), Negative predictive value 96% (73%) and Accuracy 98% (87%). A negative serologic test does not rule out the diagnosis of invasive amoebiasis. The ELISA made with antigens preserved without using enzymatic inhibitors has valuable serodiagnostic value to diagnose acute amoebic liver abscess, even in populations living in endemic zones of amoebiasis carrying antibodies against amoebas. In conclusion, ELISA-IC:MC presented better diagnostic parameters than IHA although a negative serologic test does not rule out acute invasive amoebiasis. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and amoebic liver abscess in humans, affecting millions of people worldwide. This pathogen possesses a two-stage life cycle consisting of an environmentally stable cyst and a pathogenic amoeboid trophozoite. As cysts can be ingested from contaminated food and water, this parasite is prevalent in underdeveloped countries and poses a significant health burden. Until recently there was no reliable method for inducing stage conversion in E. histolytica in vitro. As such, the reptilian pathogen, Entamoeba invadens , has long-served as a surrogate. Much remains unclear about stage conversion in these parasites and current treatments for amoebiasis are lacking, as they cause severe side effects. Therefore, new therapeutic strategies are needed. The genomes of these parasites remain enigmatic as approximately 54% of E. histolytica genes and 66% of E. invadens genes are annotated as hypothetical proteins. In this study, we characterized two hypothetical proteins in the Entamoeba species, EIN_059080, in E. invadens , and its homolog, EHI_056700, in the human pathogen, E. histolytica. EHI_056700 has no homolog in the human host. We used an RNAi-based silencing system to reduce expression of these genes in E. invadens and E. histolytica trophozoites. Loss of EIN_059080 resulted in a decreased rate of encystation and an increased rate of erythrophagocytosis, an important virulence function. Additionally, mutant parasites were more susceptible to oxidative stress. Similarly, loss of EHI_056700 in E. histolytica trophozoites resulted in increased susceptibility to oxidative stress and glucose deprivation, but not to nitrosative stress. Unlike the E. invadens mutants, E. histolytica parasites with decreased reduced expression of EHI_056700 exhibited a decreased rate of erythrophagocytosis of and adhesion to host cells. Taken together, these data suggest that these hypothetical proteins play a role in stage conversion, virulence, and the response to stress in the Entamoebae. Since parasites with reduced expression of EHI_056700 show decreased virulence functions and increased susceptibility to physiologically relevant stressors, EHI_056700 may represent a possible therapeutic target for the treatment of amoebiasis. [Display omitted] • Entamoeba genomes are mostly comprised of hypothetical proteins. • Hypothetical proteins are becoming recognized as novel drug targets. • A hypothetical protein effects stage conversion and phagocytosis in E. invadens. • A hypothetical protein effects virulence and the stress response in E. histolytica. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica programmed cell death (PCD) induced by G418 is characterized by the release of important amounts of intracellular calcium from reservoirs. Nevertheless, no typical caspases have been detected in the parasite, the PCD phenotype is inhibited by the cysteine protease inhibitor E−64. These results strongly suggest that Ca 2+ -dependent proteases could be involved in PCD. In this study, we evaluate the expression and activity of a specific dependent Ca 2+ protease, the calpain-like protease, by real-time quantitative PCR (RTq-PCR), Western blot assays and a enzymatic method during the induction of PCD by G418. Alternatively, using cell viability and TUNEL assays, we also demonstrated that the Z-Leu-Leu-Leu-al calpain inhibitor reduced the rate of cell death. The results demonstrated 4.9-fold overexpression of calpain-like gene 1.5 h after G418 PCD induction, while calpain-like protein increased almost two-fold with respect to basal calpain-like expression after 3 h of induction, and calpain activity was found to be approximately three-fold higher 6 h after treatment compared with untreated trophozoites. Taken together, these results suggest that this Ca 2+ -dependent protease could be involved in the executory phase of PCD. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatase of regenerating liver (PRL) is a group of phosphatases that has not been broadly studied in protozoan parasites. In humans, PRLs are involved in metastatic cancer, the promotion of cell migration and invasion. PTPs have been increasingly recognized as important effectors of host–pathogen interactions. We characterized the only putative protein tyrosine phosphatase PRL (PTP EhPRL) in the eukaryotic human intestinal parasite Entamoeba histolytica . Here, we reported that the EhPRL protein possessed the classical HCX 5 R catalytic motif of PTPs and the CAAX box characteristic of the PRL family and exhibited 31–32% homology with the three human PRL isoforms. In amebae, the protein was expressed at low but detectable levels. The recombinant protein (rEhPRL) had enzymatic activity with the 3- o -methyl fluorescein phosphate (OMFP) substrate; this enzymatic activity was inhibited by the PTP inhibitor o -vanadate. Using immunofluorescence we showed that native EhPRL was localized to the cytoplasm and plasma membrane. When the trophozoites interacted with collagen, EhPRL relocalized over time to vesicle-like structures. Interaction with fibronectin increased the presence of the enzyme in the cytoplasm. Using RT-PCR, we demonstrated that EhPRL mRNA expression was upregulated when the trophozoites interacted with collagen but not with fibronectin. Trophozoites recovered from amoebic liver abscesses showed higher EhPRL mRNA expression levels than normal trophozoites. These results strongly suggest that EhPRL may play an important role in the biology and adaptive response of the parasite to the host environment during amoebic liver abscess development, thereby participating in the pathogenic mechanism. [ABSTRACT FROM AUTHOR]

Rhomboid proteins represent a recently discovered family of intramembrane proteases present in a broad range of organisms and with increasing links to human diseases. The enteric parasite Entamoeba histolytica has evolved multiple mechanisms to adapt to the human host environment and establish infection. Our recent studies identified EhROM1 as a functional E. histolytica rhomboid protease with roles in adhesion to and phagocytosis of host cells. Since those studies were performed in a non-virulent strain, roles in parasite virulence could not be assessed. We focused this study on the comparison and validation of two genetic manipulation techniques: overexpression of a dominant-negative catalytic mutant of EhROM1 and knock down of EhROM1 using a RNAi-based silencing approach followed by functional studies of phenotypic analyses in virulent parasites. Both the EhROM1 catalytic mutant and parasites with EhROM1 downregulation were reduced in cytotoxicity, hemolytic activity, and directional and non-directional transwell migration. Importantly, the role for EhROM1 in cell migration mimics similar roles for rhomboid proteases from mammalian and apicomplexan systems. However, the EhROM1 catalytic mutant and EhROM1 downregulation parasites had different phenotypes for erythrophagocytosis, while complement resistance was not affected in either strain. In summary, in this study we genetically manipulated E. histolytica rhomboid protease EhROM1 by two different approaches and identified similarly attenuated phenotypes by both approaches, suggesting a novel role for EhROM1 in amebic motility. [ABSTRACT FROM AUTHOR]

Amoebiasis caused by Entamoeba histolytica continues to be one of the most common parasitic diseases in the developing world. Despite its relevance, due to the lack of accurate diagnostic methods, the true clinical and public health importance of this parasite remains uncertain. The aim of this study was to develop a new diagnostic tool to differentiate E.   histolytica from the morphologically undistinguishable E.   dispar and E.   moshkovskii . We developed a specific, fast and simple PCR-RFLP method that was able to accurately differentiate experimentally-obtained restriction patterns from the three Entamoeba species. This new method could prove useful for clinical and epidemiological purposes. [ABSTRACT FROM AUTHOR]

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1 + ) or repressed by antisense small RNA-mediated gene silencing (ICP1 − ). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N -Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1 + strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1 − strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1 + amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1 − amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis. [ABSTRACT FROM AUTHOR]

Ribosome biogenesis, a multi-step process involving transcription, modification, folding and processing of rRNA, is the major consumer of cellular energy. It involves sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica , pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. Northern hybridization from different spacer regions depicted the accumulation of unprocessed intermediates during stress. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified ribosome biogenesis factors in E. histolytica. Of the ∼279 Saccharomyces cerevisiae proteins , we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had a single copy in E. histolytica. Interestingly E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components compared to S. cerevisiae. Transcriptomic studies revealed the differential regulation of ribosomal factors both in serum starved and RRP6 down-regulation conditions. These included the NEP1 and TSR3 proteins that chemically modify 18S-rRNA. Pre-rRNA precursors accumulate upon downregulation of the latter proteins in S. cerevisiae and humans. These data reveal the major factors that regulate pre-rRNA processing during stress in E. histolytica and provide the first complete repertoire of ribosome biogenesis factors in this early-branching protist. [Display omitted] • Pre-rRNA synthesis continues, while unprocessed pre-rRNA accumulates during stress in Entamoeba histolytica. • ∼279 S. cerevisiae ribosome proteins , are represented by 188 proteins in E. histolytica. • It lacks mitochondrial ribosome biogenesis factors, has far less RNase components. • NEP1 and TSR3 proteins regulate pre-rRNA processing during growth stress in E. histolytica. • Provides the first complete repertoire of ribosome biogenesis factors in E. histolytica. [ABSTRACT FROM AUTHOR]

Highlights: [•] Overexpression of anti-Pgp5 transcript diminished P-glycoprotein expression. [•] Inhibition of PGP produced acidification of intracellular pH. [•] PGP overexpression prevent intracellular acidification and PCD. [ABSTRACT FROM AUTHOR]

Highlights: [•] We evaluated the effect of incomptine A on E. histolytica protein expression. [•] 21 proteins were differentially expressed, including three glycolytic enzymes. [•] Incomptine A also induced an increased number of glycogen granules. [•] Incomptine A could act through alteration of E. histolytica energy metabolism. [ABSTRACT FROM AUTHOR]

Abstract: Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30μg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings. [Copyright &y& Elsevier]

Highlights: [•] Entamoeba histolytica contains five genes encoding putative Ca2+-ATPases. [•] Three of the Ca2+-ATPases are grouped with plasma membrane elements (PMCA) and two with organellar members (SERCA and SPCA). [•] All Ca2+-ATPases are expressed in E. histolytica trophozoites. [•] The putative EhSERCA contains several of the structural domains of the rabbit SERCA1a. [•] The putative EhSERCA is located in a continuous cytoplasmic network and co-localized with calreticulin. [Copyright &y& Elsevier]

Highlights: [•] 2-DE and Western blot were performed on three pools of ALA pus samples. [•] Infected hamster sera and human amoebic-specific IgG were used as primary antibodies. [•] A 14kDa antigenic band was analysed by MALDI-TOF/TOF and LC–MS/MS. [•] Tyrosine kinase was the most probable identification based on the spectrum analysis. [Copyright &y& Elsevier]

Highlights: [•] Trichomonas vaginalis NADPH-dependent alcohol dehydrogenase-1 (ADH-1) reduces acetaldehyde and acetone, and oxidizes 2-propanol. [•] In addition to its canonical function, a strong reducing background activity was observed. [•] All reactions catalyzed by ADH-1 are strongly inhibited by CoA. [•] These observations also apply for the parasites Entamoeba histolytica and Tritrichomonas foetus, but not for Giardia lamblia which lacks ADH-1. [ABSTRACT FROM AUTHOR]

Abstract: It has been claimed that amoebic molecules such as amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteases are responsible for host tissue destruction and are present in both pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar. Some reports have provided evidence that after infection with E. dispar, pathological changes may occur in some humans. The aim of this study was to evaluate E. dispar pathogenicity by comparing it to the pathogenicity of E. histolytica through liver abscesses induced in hamsters. Syrian golden hamsters were challenged by intrahepatic inoculation with the 03C E. dispar strain or with two strains of E. histolytica (HM1:IMSS and EGG) to compare their virulence grades. As control groups, we used bacterial flora and Pavlova’s modified medium. Lesions were verified at 1, 3 and 6days after inoculation. Multiplex Polymerase Chain Reaction was performed to characterize each strain using EdP1/EdP2 and EhP1/EhP2 primers. The EGG and HM1:IMSS E. histolytica strains and 03C E. dispar were able to cause liver lesions. The EGG strain caused extensive hepatic abscesses, and trophozoites were found in the lesions throughout the three periods of study. The HM1:IMSS strain caused smaller abscesses when compared to EGG lesions; however, trophozoites were observed at 1 and 3days after inoculation. The 03C E. dispar strain caused intermediate abscesses when compared to the others; trophozoites were observed in all periods analyzed. The EGG strain caused progressive evolution of the injury, which differed from the HM1:IMSS and 03C strains. These results strongly suggest that the 03C E. dispar strain is pathogenic in the experimental hamster model. Additional studies are necessary to identify potential factors that regulate the manifestation of virulence of this strain and others. [Copyright &y& Elsevier]

Abstract: Vesicular trafficking, which is implicated in secretion of cytolytic molecules as well as in phagocytosis, plays an important role in the pathogenic mechanism of Entamoeba histolytica, the protozoan parasite causative of human amoebiasis. Thus, Rab GTPases, that are key regulators of vesicle trafficking, should be considered as molecules involved in the parasite virulence. EhRabB is a Rab protein located in cytoplasmic vesicles that are translocated to phagocytic mouths during ingestion of target cells, suggesting that this Rab protein is involved in phagocytosis. To prove this hypothesis, we over expressed the wild type EhrabB gene and a mutant gene encoding for a protein (RabBN118I) unable to bind guanine nucleotides and therefore constitutively inactive. The over expression of the mutated protein in E. histolytica trophozoites provoked a dominant negative effect, reflected in a significant decrease of both phagocytosis and cytopathic effect as well as in a failure to produce hepatic abscesses in hamsters. These results confirm that EhRabB is involved in phagocytosis and virulence of E. histolytica. [Copyright &y& Elsevier]

Abstract: The human cathelicidin hCAP18/LL-37 is an antimicrobial protein consisting of a conserved N-terminal prosequence called the cathelin-like domain and a C-terminal peptide called LL-37. This peptide contains 37 amino acid residues, and several truncated variants obtained from natural sources or by chemical synthesis differ in their capability to damage Gram positive and Gram negative bacteria as well as Candida albicans. KR-12 is the shortest peptide (12 amino acids) of LL-37 that has conserved antibacterial activity. In addition to LL-37, other active cathelicidin-derived peptides have been reported; for instance, the peptides KR-20, a 20-aa derivative of LL-37, and KS-30, a 30-aa derivative of LL-37, have been found in human sweat. Both peptides exhibit an overall increased antibacterial and antifungal activity when compared with LL-37. We investigated the effect of LL-37 and three peptides derived from this antimicrobial molecule, KR-12, KR-20 and KS-30, on the integrity of Entamoeba histolytica trophozoites. The four peptides showed effects on E. histolytica integrity and viability in the concentration range of 10–50μM. The peptides KR-12, KR-20, KS-30 and LL-37 differed in their capability to damage the parasite integrity, with KR-20 being the most effective and with KR-12 and LL-37 being less active. These results demonstrate the ability of antimicrobial peptides derived from human cathelicidin to damage Entamoeba trophozoites. Moreover, it was shown that the integrity of the peptides is altered in the presence of an ameba soluble fraction with cysteine protease activity. [Copyright &y& Elsevier]

Abstract: Entamoeba histolytica is among the most deadly parasites accounting for the second highest mortality rate among parasitic diseases. Nevertheless, contrary to trypanosomatids, this protozoan in hardly studied by parasitology groups. This astonishing discrepancy is largely due to the remarkable intricate conditions required for parasite proliferation in vitro, particularly whenever large cell numbers are required. The present study was undertaken in order to optimize E. histolytica culturing harvest, using mineral oil layers preventing culture medium-air contact to maintain anaerobic conditions in culture plate wells. 2×104 trophozoites were plated on each well in 2.0mL YI-S-33 medium, supplemented with bovine serum and 700μL mineral oil. Parasites were daily quantified by light microscopy counting for up to 96h and trophozoite motility was also assessed. We notice that E. histolytica cultures in 24-well plates reached several-fold higher cell densities, particularly whenever the mineral oil layer was placed on top of the medium surface, blocking the air interface. At least 99% of the parasites were vigorously motile for 72h in oil-containing wells, whereas only less than 5% displayed significant motility in oil-devoid wells. In order to determine whether such different growth responses were due at least in part to the oxidative stress, we used the reactive oxidant species fluorescent probe dihydroethidium (DHE). The remarkably higher DHE parasite labeling in oil-devoid cultures indicate that oxidative stress reduction can play a significant role in elevated growth rates observed in oil supplemented cultures. Propidium iodide and Trypan blue dye-exclusion assays indicate that parasite necrosis resulted from the stressing conditions. The present study indicates that E. histolytica culturing in oil-sealed wells may comprise a valuable tool for bioactivity of antiparasitic compounds. [Copyright &y& Elsevier]

Abstract: Precision-cut liver slices (PCLS) are mainly used to evaluate hepatotoxicity and metabolism of chemicals, as well as to study mechanisms of liver damage and repair. However, recently they have been used as a system to study amoebic infections. The aim of this study was to validate this model as an alternative for experimental amoebic liver absess (ALA) in animals. To do this, the PCLS was analyzed for the expression of amoebapore and cysteine proteinases 1 and 5, three of the most studied virulence factors of Entamoeba histolytica, as well as the induction of apoptosis and cytokines production in response to the ex vivo infection. PCHLS were prepared with the Brendel-Vitron tissue slicer and then, infected with 200,000 trophozoites of E. histolytica. Samples were taken at 0, 6, 12, 18, and 24h and compared to control non-infected slices. Morphological studies were performed in order to verify the infection; while apoptosis was studied by TUNEL and PAS techniques. The expression of cysteine proteinases (1 and 5), and amoebapore, was analyzed by real-time PCR. By using ELISA assays, the production of cytokines was also studied. PCHLS were found to be a reproducible infection system, and E. histolytica caused the expression of cysteine proteinases and amoebapore in infected slices. At the same time, trophozoites induce release of cytokines and apoptotic death of the hepatocytes close to them. PCHLS represent a new and suitable alternative model to study the pathogenesis of hepatic amoebiasis. [Copyright &y& Elsevier]

Abstract: Cysteine proteinases 4 (EhCP4) of Entamoeba histolytica are considered important for ameba pathogenicity. The recombinant gene was obtained by cloning and expression of the EhCP4 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig–E. histolytica model. There was a 53.16% reduction (P <0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP4 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P <0.001). This is a first report demonstrating that a recombinant form of EhCP4 generated in E. coli, to immunize a minipig model of E. histolytica, and there is significant protection. This study may help to understand the EhCP4 for human in the future. [Copyright &y& Elsevier]

Abstract: Entamoeba histolytica cysteine proteinase gene 5(EhCP5) is one of the major proteinase genes of all EhCP-transcripts. The amebiasis cysteine proteinase gene encoding an antigen from E. histolytica, as well as the recombinant EhCP5, obtained by cloning and expression of the EhCP5 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in Minipig against challenge infection in a minipig-E. histolytica model. There was a 52.27% reduction (P <0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-EhCP5 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P <0.0001). Our data will help to know the mechanism of vaccinal protection of E. histolytica. [Copyright &y& Elsevier]

Abstract: The amebiasis cysteine proteinase gene (ACP1) encoding an antigen from Entamoeba histolytica, as well as the recombinant ACP1, obtained by cloning and expression of the ACP1 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in minipig against challenge infection in a minipig –E. histolytica model. There was a 64.52% reduction (P <0.001) in the group of recovery of challenged E. histolytica compared with that in the control group. Specific anti-ACP1 antibodies from immune protected minipig had significantly higher levels of immunoglobulin G (IgG) (P <0.001). Our data indicate recombinant ACP1 may be a potential target as a vaccine antigen. [Copyright &y& Elsevier]

Abstract: The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process. [Copyright &y& Elsevier]

Abstract: Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. Here, we report the biochemical characterization of a PDI enzyme from the protozoan parasite Entamoeba histolytica (EhPDI). Our results show that EhPDI behaves mainly as an oxidase/isomerase and can be inhibited by bacitracin, a known PDI inhibitor; moreover, it exhibits chaperone-like activity. Albeit its physiological role in the life style of the parasite (including virulence and survival) remains to be studied, EhPDI could represent a potential drug target for anti-amebic therapy. [Copyright &y& Elsevier]

Abstract: Total nucleic acids from sporulated oocysts of Eimeria tenella isolated from Changchun in China were found to contain three extrachromosomal double-stranded RNA segments (dsRNAs) of 1.4, 2.4 and 3.6kb in sizes. These RNAs were resistant to RNase A digestion under high salt concentration (0.3M NaCl). RNA-dependent RNA polymerase (RDRP) activity was detected in crude extracts of E. tenella sporulated oocysts containing these nucleic acid species. Virus-like particles (VLPs) were shown to have a diameter of approximately 38nm under Electron Microscopy (EM) after purification by sucrose density gradient centrifugation. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this isolate as E. tenella virus (ETV) which is the first virus isolated from E. tenella. [Copyright &y& Elsevier]

Abstract: The influence of low-frequency electromagnetic (LF-EM) waves on microorganisms has been a subject of experimental investigations for more than two decades and the results are promising. In parallel, an interesting procedure known as biophysical-information-therapy or bioresonance therapy (BRT) which in principle is based on LF-EM stimulation, has emerged. BRT was discovered in the late 1980’s but it is still poorly studied. This paper demonstrates that by transferring metronidazole information to water samples by an electronic amplifier (BRT device), the growth of axenically cultured trophozoites of Entamoeba histolytica and Trichomonas vaginalis is significantly inhibited, compared with those cultures treated with non and sham electro-transferred water samples. A positive control of metronidazole, a well-known cytotoxic drug against parasites, was used as a reference. [ABSTRACT FROM AUTHOR]

Abstract: We have demonstrated that programmed cell death (PCD) in Entamoeba histolytica is induced in vitro by G418 aminoglycoside antibiotic. To ascertain if biochemical and morphological changes previously observed are paired to molecular changes that reflect a genetic program, we looked here for early differential gene expression during the induction of PCD. Using cDNA-amplified fragment length polymorphisms (AFLPs) and in silico derived analysis we showed in E. histolytica a differential gene expression during PCD induced by G418. The genes identified encoded for proteins homologous to Glutaminyl-tRNA synthase, Ribosomal Subunit Proteins 40S and 18S, Saposin-like, Silent Information Regulator-2 (Sir-2), and Grainins 1 and 2. Using real-time quantitative PCR (RT Q-PCR), we found that glutaminyl-tRNA synthetase, sir-2, grainins and saposin-like genes were strongly overexpressed after 30min of PCD induction, while its expression dramatically decreased up to 60min. On the other hand, overexpression of ribosomal genes increased only 7-fold of basal expression, showing a progressive down-regulation up to 90min. glutaminyl-tRNA synthetase, sir-2 and grainins could act as negative regulators of PCD, trying to control the biochemical changes related to PCD activation. Overexpression of saposin-like gene could act as up-regulator of some cell death pathways. Our results give evidence of the first genes identified during the early stage of PCD in E. histolytica that could be implicated in regulation of apoptotic pathways. [ABSTRACT FROM AUTHOR]

Abstract: Amebiasis is the disease caused by the enteric dwelling protozoan parasite Entamoeba histolytica. The WHO considers amebiasis as one of the major health problems in developing countries; it is surpassed by only malaria and schistosomiasis for death caused by parasitic infection. E. histolytica primarily lives in the colon as a harmless commensal, but is capable of causing devastating dysentery, colitis and liver abscess. What triggers the switch to a pathogenic phenotype and the onset of disease is unknown. We are becoming increasingly aware of the complexity of the host–parasite interaction. During chronic stages of amebiasis, the host develops an immune response that is incapable of eliminating tissue resident parasites, while the parasite actively immunosuppresses the host. However, most individuals with symptomatic infections succumb only to an episode of dysentery. Why most halt invasion and a minority progress to chronic disease remains poorly understood. This review presents a current understanding of the immune processes that shape the outcome of E. histolytica infections during its different stages. [Copyright &y& Elsevier]

Abstract: Entamoeba histolytica antigens recognized by salivary IgA from infected patients include the 29kDa antigen (Eh29), an alkyl hydroperoxide reductase. Here, we investigate the potential of recombinant Eh29 and an Eh29-cholera toxin subunit B (CTxB) fusion protein to confer protection against intestinal amoebiasis after oral immunization. The purified Eh29–CTxB fusion retained the critical ability to bind ganglioside GM1, as determined by ELISA. Oral immunization of C3H/HeJ mice with Eh29 administered in combination with a subclinical dose of whole cholera toxin, but not as an Eh29–CTxB fusion, induced elevated levels of intestinal IgA and serum IgG anti-Eh29 antibodies that inhibited trophozoites adherence to MDCK cell monolayers. The 80% of immunized mice seen to develop IgA and IgG immune responses showed no evidence of infection in tissue sections harvested following intracecal challenge with virulent E. histolytica trophozoites. These results suggest that Eh29 is capable of inducing protective anti-amoebic immune responses in mice following oral immunization and could be used in the development of oral vaccines against amoebiasis. [Copyright &y& Elsevier]

Abstract: Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses. [ABSTRACT FROM AUTHOR]

Abstract: Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5–96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg2+, Mn2+, or Co2+, and inhibited by Zn2+, Hg2+, Ca2+, and EDTA. SMase-C was active in the pH range of 3–10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence. [Copyright &y& Elsevier]

Abstract: A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg2+-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn2+ and partially inhibited by Zn2+. nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5mM Mn2+, and abolished by 5mM Zn2+. A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35–37kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined. [Copyright &y& Elsevier]

Abstract: We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG’s anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh. [Copyright &y& Elsevier]

Abstract: Despite their well recognized importance in pathogenesis of Entamoeba histolytica there are few studies dealing with the assembly and secretion of glycoproteins that participate in the adhesion to target cells and in the dissemination of the parasite in infected tissues. Some of these studies refer to the identification and, in some cases, the characterization of glycosyl transferases and glycosidases involved in the biosynthesis of these macromolecules as well as to compartments involved in the amoeba dolichol-linked glycosylation pathway. While an N-glycan trimming α-mannosidase has been demonstrated in E. histolytica, little is known on its cellular distribution and properties. Here we describe the presence and partial biochemical characterization of soluble and MMF-associated forms of α-mannosidase and the separation of at least three internal membrane structures enriched with this glycosidase. Results are discussed in terms of the possible identity of α-mannosidase activity and the potential precursor–product relationship between the two enzyme forms. [Copyright &y& Elsevier]

Entamoeba histolytica, the causative agent of amebiasis infects through its cyst form and this transmission may be blocked using encystation specific protein as drug target. In this study, we have characterized the enzyme chitinase which express specifically during encystation. The reptilian parasite Entamoeba invadens, used as a model for encystation study contain three chitinases. We report the molecular cloning, over-expression and biochemical characterization of all three E. invadens chitinase. Cloned chitinases were over-expressed in bacterial system and purified by affinity chromatography. Their enzymatic profiles and substrate cleaving patterns were characterized. All of them showed binding affinity towards insoluble chitin though two of them lack the chitin binding domain. All the chitinases cleaved and released dimmers from the insoluble substrate and act as an exochitinase. Homology modeling was also done to understand the substrate binding and cleavage pattern. [Copyright &y& Elsevier]

Abstract: Previous studies from this laboratory have dealt with the purification and biochemical characterization of ornithine decarboxylase (ODC) from Entamoeba histolytica. Enzyme compartmentalization has been described as a major mechanism in the regulation of polyamine metabolism. However, the subcellular location of ODC in the human parasite has remained unresolved. To examine this issue, we cloned the full-length gene (Ehodc) encoding for the parasite enzyme, whose open reading frame encodes for a peptide of 412 amino acids with an estimated molecular mass of 46kDa that exhibits similarity to other ODCs. Heterologous overexpression of the gene allowed us to purify the recombinant protein (rEhODC) by metal affinity chromatography. The purified polypeptide was used to raise heteroclonal antibodies that were utilized to localize the enzyme in situ by immunofluorescence and confocal microscopy. EhODC was observed to be associated with the plasma membrane, in vesicles close to the plasma membrane and in the EhkOs organelle. [Copyright &y& Elsevier]

Abstract: Cysteine proteinase (CP) activity and CP5 mRNA levels were analyzed in eleven samples of Entamoeba histolytica isolated from patients presenting different clinical profiles. The virulence degree of the isolates, determined in hamster liver, correlated well with the clinical form of the patient and culture conditions. CP5 mRNA levels were also determined in sample freshly picked up directly from liver amoebic abscess. Differences were not observed in the levels of CP5 mRNA and CP specific activity among the cultured samples. However, different levels of CP5 mRNA were observed in trophozoite freshly isolated from hepatic amoebic lesions. These results reinforce the importance of CP5 for the virulence of amoebae and the need for studies with the parasite present in lesions to validate mechanisms involved in pathogenesis of amoebiasis. [Copyright &y& Elsevier]

Abstract: Monoxenic cultivation of pathogenic Entamoeba histolytica trophozoites with Escherichia coli serotype 055 which binds strongly to the Gal/GalNAc amoebic lectin, markedly improved the growth of E. histolytica and produced a significant decrease in cysteine proteinase activity and a lower cytopathic activity on monolayer cells after 3 months of monoxenic culture. However, after long term monoxenic culture (12 months) the proteolytic and cytopathic activities were recovered and the amoebic growth reached the maximum yield. Employing the GeneFishingR technology and DNA macroarrays we detected differentially gene expression related to the amoebic interaction with bacteria. A number of differentially expressed genes encoding metabolic enzymes, ribosomal proteins, virulence factors and proteins related with cytoskeletal and vesicle trafficking were found. These results suggest that E. coli 055 has a nutritional role that strongly supports the amoebic growth, and is also able to modulate some biological activities related with amoebic virulence. [Copyright &y& Elsevier]