Entamoeba histolytica: differences in phagosome acidification and degradation between attenuated and virulent strains

Phagocytosis is the important virulent determinant of the enteric protozoan parasite Entamoeba histolytica. We compared the kinetics of phagosome maturation of attenuated and highly-virulent strains of E. histolytica using video microscopy. Phagosomes of attenuated strains were acidified rapidly within 2 min after phagosome formation (at the rate of 0.96 pH/min), persisted at pH 4.46+/-0.13, and degraded ingested GFP-Leishmania very efficiently (90-94% GFP fluorescence was lost in 30 min), while phagosomes of highly-virulent strains were acidified slowly (0.69 pH/min), persisted at 5.11+/-0.23, and degraded GFP less efficiently (60-71% decrease). These results suggest that efficiency of phagosome maturation is most probably inversely correlated with apparent virulence.     

Entamoeba histolytica: alterations in EhRabB protein in a phagocytosis deficient mutant correlate with the Entamoeba dispar RabB sequence

We analyzed the expression and location of EhRabB in clone L-6, a phagocytosis-deficient mutant of Entamoeba histolytica, in comparison with the wild-type clone A. Intriguingly, trophozoites of clone L-6 express more EhRabB than those of clone A. However, the majority of EhRabB-containing vesicles remained in the cytoplasm of clone L-6 during phagocytosis. To investigate molecular alterations in EhRabB of clone L-6 we compared the EhrabB gene sequences from clones L-6 and A. We also isolated, sequenced and compared the RabB protein of Entamoeba dispar. Results showed that EhrabB gene of clone L-6 is 98.2 and 94.1% identical to rabB genes of E. dispar and clone A, respectively. The rabB genes from clone A and E. dispar have 92.2% identity. Four out of five amino acids changes in RabB proteins of clone L-6 and E. dispar are shared. These changes may alter the binding of effector proteins and the specific subcellular location of EhRabB.     

Entamoeba histolytica: fibrilar aggregates in dividing trophozoites

Entamoeba histolytica trophozoite cytokinesis is dependent upon cytoskeletal elements such as filamentous actin and myosin. Here we present confocal and transmission electron microscopy studies of this process. A sequence in the formation of the contractile ring was shown with rhodamine-phalloidine staining. Ultrastructural analysis revealed the presence of fibrilar aggregates in the cytoplasm of dividing trophozoites. Among them two filaments of different diameter were identified. These aggregates presented repeating assemblies of thin and thick filaments that in cross section revealed a muscle-like appearance. Our results suggest that these aggregates constitute the contractile ring responsible for the separation of daughter cells.     

Entamoeba histolytica: Cysteine proteinase activity and virulence. Focus on cysteine proteinase 5 expression levels

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.     

Inhibition of gene expression with double strand RNA interference in Entamoeba histolytica

In order to inhibit gene expression in Entamoeba histolytica, we have developed a method based on expressing double strand RNA interference constructs in stable transformants. The 5' end of Eh Dia was cloned head to head with an intervening non-specific stuffer fragment in the E. histolytica expression vector pJST4. This construct was transformed in E. histolytica HM1:IMSS trophozoites and stable transformants were selected with 20microg/ml G418. Our results show that expression of Eh Dia was completely inhibited in these transformants. These stable transformants could be maintained indefinitely without expression of Eh Dia. This method therefore provides an effective tool to study the phenotypic changes, which occur due to inhibition of gene expression in the absence of mutants and other microbiological manipulations in this protozoan parasite.     

Purification and biochemical characterisation of a membrane-bound alpha-glucosidase from the parasite Entamoeba histolytica

An alpha-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-alpha-D-glucopyranoside (MUalphaGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 degrees C. Although the enzyme preferentially hydrolysed nigerose (alpha1,3-linked), it also cleaved kojibiose (alpha1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (alpha1,4), trehalose (alpha1,1) and isomaltose (alpha1,6). Activity on alpha1,3- and alpha1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy-D-glucose and 6-deoxy-D-glucose were strong inhibitors of activity, whereas 2-deoxy-D-glucose and other monosaccharides had no effect. Enzyme activity on MUalphaGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)-N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.     

Cloning and partial characterization of Entamoeba histolytica PTPases

Reversible protein tyrosine phosphorylation is an essential signal transduction mechanism that regulates cell growth, differentiation, mobility, metabolism, and survival. Two genes coding for protein tyrosine phophatases, designed EhPTPA and EhPTPB, were cloned from Entamoeba histolytica. EhPTPA and EhPTPB proteins showed amino acid sequence identity of 37%, both EhPTPases showed similarity with Dictyostelium discoideum and vertebrate trasmembranal PTPases. mRNA levels of EhPTPA gene are up-regulated in trophozoites recovered after 96h of liver abscess development in the hamster model. EhPTPA protein expressed as a glutathione S-transferase fusion protein (GST::EhPTPA) showed enzymatic activity with p-nitrophenylphosphate as a substrate and was inhibited by PTPase inhibitors vanadate and molybdate. GST::EhPTPA protein selectively dephosphorylates a 130kDa phosphotyrosine-containing protein in trophozoite cell lysates. EhPTPA gene codifies for a 43kDa native protein. Up-regulation of EhPTPA expression suggests that EhPTPA may play an important role in the adaptive response of trophozoites during amoebic liver abscess development.     

Entamoeba histolytica and Entamoeba dispar: prevalence infection in a rural Mexican community

The frequency of Entamoeba histolytica and Entamoeba dispar infection was analyzed in a rural community in the state of Morelos, Mexico, through PCR technique by using specie specific primer. The E. histolytica specie was detected in 33 of 290 analyzed stool samples (11.4%), E. dispar specie was observed in 21 samples (7.2%) and both species of Entamoeba were detected in seven samples (2.4%). So a higher E. histolytica than E. dispar frequency infection was detected (13.8 versus 9.6%). Even though in our design we did not considered the follow-up of included individuals, the absence of invasive amebiasis cases in the studied population during our stay in town was unexpected.     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.     

Identification of EhICP1, a chagasin-like cysteine protease inhibitor of Entamoeba histolytica

Based on the Entamoeba histolytica genome project (www.sanger.ac.uk/Project/E_histolytical/) we have identified a cysteine protease inhibitor, EhICP1 (amoebiasin 1), with significant homology to chagasin. Recombinant EhICP1 inhibited the protease activity of papain and that of a trophozoite lysate with Ki's in the picomolar range. By immunocytology, we localized the endogenous approximately 13 kDa EhICP1 in a finely dotted subcellular distribution discrete from the vesicles containing the amoebic cysteine protease, EhCP1 (amoebapain). In an overlay assay, we observed binding of recombinant EhICP1 to EhCP1. As a heptapeptide (GNPTTGF) corresponding to the second conserved chagasin motif inhibited the protease activity of both papain (K) 1.5 microM) and trophozoite extract (Ki in sub-mM range), it may be a candidate for the rational development of anti-amoebiasis drugs.     

Redox regulation of UDP-glucose pyrophosphorylase from Entamoeba histolytica

Amoebiasis is an intestinal infection caused by the human pathogen Entamoeba histolytica and representing the third leading cause of death by parasites in the world. Host-parasite interactions mainly involve anchored glycoconjugates localized in the surface of the parasitic cell. In protozoa, synthesis of structural oligo- and polysaccharides occurs via UDP-glucose, generated in a reaction catalyzed by UDP-glucose pyrophosphorylase. We report the molecular cloning of the gene coding for this enzyme from genomic DNA of E. histolytica and its recombinant expression in Escherichia coli cells. The purified enzyme was kinetically characterized, catalyzing UDP-glucose synthesis and pyrophosphorolysis with V_max values of 95 U/mg and 3 U/mg, respectively, and affinity for substrates comparable to those found for the enzyme from other sources. Enzyme activity was affected by redox modification of thiol groups. Different oxidants, including diamide, hydrogen peroxide and sodium nitroprusside inactivated the enzyme. The process was completely reverted by reducing agents, mainly cysteine, dithiothreitol, and thioredoxin. Characterization of the enzyme mutants C94S, C108S, C191S, C354S, C378S, C108/378S, M106S and M106C supported a molecular mechanism for the redox regulation. Molecular modeling confirmed the role of specific cysteine and methionine residues as targets for redox modification in the entamoebic enzyme. Our results suggest that UDP-glucose pyrophosphorylase is a regulated enzyme in E. histolytica. Interestingly, results strongly agree with the occurrence of a physiological redox mechanism modulating enzyme activity, which would critically affect carbohydrate metabolism in the protozoon.     

Structure and inactivation of triosephosphate isomerase from Entamoeba histolytica

Triosephosphate isomerase (TIM) has been proposed as a target for drug design. TIMs from several parasites have a cysteine residue at the dimer interface, whose derivatization with thiol-specific reagents induces enzyme inactivation and aggregation. TIMs lacking this residue, such as human TIM, are less affected. TIM from Entamoeba histolytica (EhTIM) has the interface cysteine residue and presents more than ten insertions when compared with the enzyme from other pathogens. To gain further insight into the role that interface residues play in the stability and reactivity of these enzymes, we determined the high-resolution structure and characterized the effect of methylmethane thiosulfonate (MMTS) on the activity and conformational properties of EhTIM. The structure of this enzyme was determined at 1.5A resolution using molecular replacement, observing that the dimer is not symmetric. EhTIM is completely inactivated by MMTS, and dissociated into stable monomers that possess considerable secondary structure. Structural and spectroscopic analysis of EhTIM and comparison with TIMs from other pathogens reveal that conformational rearrangements of the interface after dissociation, as well as intramonomeric contacts formed by the inserted residues, may contribute to the unusual stability of the derivatized EhTIM monomer.     

Differentiation of Entamoeba histolytica: a possible role for enolase

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 48 kDa 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.     

Cyclosporin A inhibits calcineurin (phosphatase 2B) and P-glycoprotein activity in Entamoeba histolytica

Cyclosporin A (CsA) inhibits the proliferation of several protozoan parasites through blocking the activity of calcineurin (Cn) or P-glycoproteins (Pgp). We report here, that inhibition of the proliferation of Entamoeba histolytica trophozoites, the causal agent of human amebiasis, is due to interference of the phosphatase activity of Cn, in a similar fashion to the effect of this immunosuppressive drug on T lymphocytes. The non-immunosuppressive CsA analog PSC-833, which binds Pgp without interfering the function of Cn, did not inhibit the proliferation of HM1:IMSS trophozoites. Moreover, phosphatase activity of amebic Cn, detected using the phosphopeptide RII, was drastically affected by incubation with CsA, but not with PSC-833. On the other hand, both drugs were also tested on clone C2 trophozoites, which grow in the presence of emetine due to over-expression of Pgp. The effect of CsA was similar to that observed on HM1:IMSS trophozoites, whereas PSC-833 only affected the proliferation and viability of clone C2 when the trophozoites were grown in the presence of 40 microM of emetine, suggesting an interference of the Pgp activity. This suggestion was confirmed by results from experiments of Pgp-dependent effux of rhodamine from pre-loaded trophozoites, in the presence of either of these drugs. Therefore, CsA inhibition of E. histolytica trophozoite proliferation is more likely due to Cn than Pgp activity inhibition.     

Inhibitors of Escherichia coli serine acetyltransferase block proliferation of Entamoeba histolytica trophozoites

The protozoan parasite Entamoeba histolytica is the etiologic agent of amebiasis, a major global public health problem, particularly in developing countries. There is an effective anti-amoebic drug available, however its long term use produces undesirable side effects. As E. histolytica is a micro-aerophilic organism, it is sensitive to high levels of oxygen and the enzymes that are involved in protecting against oxygen-stress are crucial for its survival. Therefore serine acetyltransferase, an enzyme involved in cysteine biosynthesis, was used as a target for identifying potential inhibitors. Virtual screening with Escherichia coli serine acetyltransferase was carried out against the National Cancer Institute chemical database utilizing molecular docking tools such as GOLD and FlexX. The initial analysis yielded 11 molecules of which three compounds were procured and tested for biological activity. The results showed that these compounds partially block activity of the E. coli enzyme and the growth of E. histolytica trophozoites but not mammalian cells.     

Effect of iron on adherence and cytotoxicity of Entamoeba histolytica to CHO cell monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.     

Entamoeba histolytica: the serine-rich gene polymorphism-based genetic variability of clinical isolates from Georgia

It is generally accepted that a majority of individuals infected by Entamoeba histolytica do not develop symptomatic disease. However, the parasite and the host factors contributing to the development of the disease, remain undetermined. It is also unclear why certain individuals develop extra-intestinal amebiasis without exhibiting apparent intestinal symptoms. An outbreak of amebic liver abscess in Tbilisi, Georgia in 1998-1999 suggested that the causative E. histolytica strain had an unusual propensity for extra-intestinal spread. To correlate the genetic differences with pathogenic potential of the parasite, we have examined the SREHP gene polymorphisms among Georgian E. histolytica isolates. Comparison of polymorphic patterns revealed the presence of several different genotypes of E. histolytica, thus preventing an association of a single genotype with hepatic disease, but supporting the previous finding of extensive genetic diversity among E. histolytica isolates from the same geographic origin.     

PCR diagnosis of Entamoeba histolytica cysts in stool samples

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.