Involvement of Src family tyrosine kinase in apoptosis of human neutrophils induced by protozoan parasite Entamoeba histolytica

Tyrosine kinases are one of the most important regulators for intracellular signal transduction related to inflammatory responses. However, there are no reports describing the effects of tyrosine kinases on neutrophil apoptosis induced by Entamoeba histolytica. In this study, isolated human neutrophils from peripheral blood were incubated with live trophozoites in the presence or absence of tyrosine kinase inhibitors. Entamoeba-induced receptor shedding of CD16 and PS externalization in neutrophils were inhibited by pre-incubation of neutrophils with the broad-spectrum tyrosine kinase inhibitor genistein or the Src family kinase inhibitor PP2. Entamoeba-induced ROS production was also inhibited by genistein or PP2. Moreover, genistein and PP2 blocked the phosphorylation of ERK and p38 MAPK in neutrophils induced by E. histolytica. These results suggest that Src tyrosine kinases may participate in the signaling event for ROS-dependent activation of MAPKs during neutrophil apoptosis induced by E. histolytica.     

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: 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.     

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.     

Characterization of YS-27, an axenic Korean strain of Entamoeba histolytica

Characterization of YS-27, an axenic Entamoeba strain, was performed by three different laboratory methods. Zymodeme analysis using starch gel electrophoresis and PCR with species-specific primers showed that YS-27 is a pathogenic Entamoeba which belongs to the group II zymodeme. Pathogenicity of YS-27 was further confirmed by observing the formation of liver abscess in Mongolian gerbils. These results showed that YS-27 is E. hisolytica.     

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.     

Inhibitory effects of Iranian Thymus vulgaris extracts on in vitro growth of Entamoeba histolytica

One of the most common drugs used against a wide variety of anaerobic protozoan parasites is metronidazole. However, this drug is mutagenic for bacteria and is a potent carcinogen for rodents. Thymus vulgaris is used for cough suppression and relief of dyspepsia. Also it has antibacterial and antifungal properties. The aim of this study was to investigate antiamebic effect of Thymus vulgaris against Entamoeba histolytica in comparison with metronidazole. One hundred gram air-dried T. vulgaris plant was obtained and macerated at 25 degrees C for 14 days using n-hexane and a mixture of ethanol and water. For essential oil isolation T. vulgaris was subjected to hydrodistillation using a clevenger-type apparatus for 3 hr. E. histolytica, HM-1: IMSS strain was used in all experiments. It was found that the minimal inhibitory concentration (MIC) for T. vulgaris hydroalcoholic, hexanic extracts, and the essential oil after 24 hr was 4 mg/mL, 4 mg/mL, and 0.7 mg/mL, respectively. After 48 hr the MIC for T. vulgaris hydroalcoholic and hexanic extracts was 3 and 3 mg/mL, respectively. Therefore, it can be concluded that the Iranian T. vulgaris is effective against the trophozoites of E. histolytica.     

Calpain-dependent calpastatin cleavage regulates caspase-3 activation during apoptosis of Jurkat T cells induced by Entamoeba histolytica

In this study, we investigated whether there is a signalling interaction between calpain and caspase-3 during apoptosis in Jurkat T cells by Entamoeba histolytica. When Jurkat cells were co-incubated with E. histolytica, phosphatidylserine externalisation and DNA fragmentation markedly increased compared with results for cells incubated with medium alone. In addition, E. histolytica strongly induced cleavage of caspases-3, -6, -7 and poly(ADP-ribose) polymerase. A rise in intracellular calcium levels and activation of calpain were seen in Jurkat cells after exposure to E. histolytica. Pretreatment of Jurkat cells with calpain inhibitor calpeptin effectively blocked E. histolytica-triggered cleavage of caspase-3 as well as calpain. In contrast, pan-caspase inhibitor did not affect E. histolytica-induced calpain activation. In addition, incubation with E. histolytica resulted in multiple fragmented bands of calpastatin, which is an endogenous inhibitor of calpain, in Jurkat T cells. Moreover, Entamoeba-induced calpastatin degradation was dramatically suppressed by pretreatment with calpeptin, but not by z-VAD-fmk. Entamoeba-induced DNA fragmentation was strongly retarded by z-VAD-fmk, but not calpeptin. Our results suggest that calpain-mediated calpastatin degradation plays a crucial role in regulation of caspase-3 activation during apoptosis of Jurkat T cells by E. histolytica.     

The diversity of Rab GTPases in Entamoeba histolytica

Rab proteins are ubiquitous small GTP-binding proteins that form a highly conserved family and regulate vesicular trafficking. Recent completion of the genome of the enteric protozoan parasite Entamoeba histolytica enabled us to identify an extremely large number (>90) of putative Rab genes. Multiple alignment and phylogenic analysis of amebic, human, and yeast Rab showed that only 22 amebic Rab proteins including EhRab1, EhRab2, EhRab5, EhRab7, EhRab8, EhRab11, and EhRab21 showed significant similarity to Rab from other organisms. The 69 remaining amebic Rab proteins showed only moderate similarity (<40% identity) to Rab proteins from other organisms. Approximately one-third of Rab proteins including Rab7, Rab11, and RabC form 15 subfamilies, which contain up to nine isoforms. Approximately 70% of amebic Rab genes contain single or multiple introns, and this proportion is significantly higher than that of common genes in this organism. Twenty-five Rabs possess an atypical carboxyl terminus such as CXXX, XCXX, XXCX, XXXC, and no cysteine. We propose annotation of amebic Rab genes and discuss biological significance of this extraordinary diversity of EhRab proteins in this organism.     

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.     

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.     

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.     

The MAK16 Gene of Entamoeba histolytica and Its Identification in Isolates from Patients

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher''s exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient''s symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.     

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.     

Characterization of a novel Entamoeba histolytica strain from Burkina Faso, Africa, possessing a unique hexokinase-2 gene

An Entamoeba histolytica strain (BF-841 cl1) that originated from Burkina Faso, Africa presented with novel, polymorphic genotypes of the serine-rich E. histolytica protein and the anodic hexokinase-2 (HXK-2) isoenzyme band, which showed less electrophoretic mobility than that of an E. histolytica reference strain [HM-1:IMSS cl6 (zymodeme (Z)-II)] by starch gel electrophoresis and isoelectric focusing (IEF). The HXK-2 gene of BF-841 cl1 had amino acid variations at four positions compared to the sequence of HM-1: IMSS cl6. These variations were absent from the sequences of four other E. histolytica strains with different zymodemes [KU27 (Z-II), SAW1627 (Z-IIα-), SAW755CR clB (Z-XIV), and KU2 (Z-XIX)]. The results of IEF showed no difference in the substrate specificity of HXK (HXK-1 and HXK-2) between BF-841 cl1 and the three reference E. histolytica strains (HM-1:IMSS cl6, SAW755 clB, and KU27). It was also confirmed that BF-841 cl1 was able to form liver abscesses in Syrian hamsters.     

Entamoeba histolytica: genetic diversity of African strains based on the polymorphism of the serine-rich protein gene

The polymorphism of the serine-rich Entamoeba histolytica protein (SREHP) among isolates obtained from different geographic regions was analyzed by a nested PCR followed by restriction analysis. Thirteen different profiles were generated from 23 E. histolytica isolates from Cameroon, Zimbabwe and South Africa while 20 others were generated from 38 E. histolytica PCR positive stool samples from South Africa. One of the profiles was common to isolates from Cameroon, Zimbabwe and South Africa and constituted the most prevalent (26.1%) of all the profiles. However, profiles unique to each country were also observed amongst the samples. A non-significant difference was observed between isolates from diarrheic and non-diarrheic samples. Of interest, of the five HIV positive stool samples three had the same profile indicating the possibility that some E. histolytica strains might be more common/pathogenic in immuno-compromised individuals. The results obtained showed that African isolates of E. histolytica may possess extremely complex genetic structures independent of geographic location. This study indicates that certain profiles might be responsible for the presentation of intestinal amoebic symptoms. However, more extended studies need to be performed in order to confirm these observations.     

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.     

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.