Introns of Entamoeba histolytica and Entamoeba dispar

The genome of Entamoeba histolytica is considered to possess very few intervening sequences (introns), as only 5 intron-containing genes from this protozoan parasite have been reported so far. However, while sequencing a number of genomic contigs as well as three independent genes coding for ribosomal protein L27a, we have identified 9 additional intron-containing genes of E. histolytica and the closely related species Entamoeba dispar,indicating that introns are more common in these organisms than previously suggested. The various amoeba introns are relatively short comprising between 46 and 115 nucleotides only and have a higher AT-content compared to thecorresponding exon sequences. In contrast to higher eukaryotes, amoeba introns do not contain a well-conserved branch point consensus, and have extended donor and acceptor splice sites of the sequences GTTTGTT and TAG, respectively. Consistent with the close phylogenetic relationship of E. histolytica and E. dispar, the position and length of introns is conserved between the two species but the degree of sequence identity is reduced compared to orthologous coding regions.     

Comparison of adherence, cytotoxicity, and Gal/GalNAc lectin gene structure in Entamoeba histolytica and Entamoeba dispar

The recent development of axenic culture for E. dispar allowed us to examine this ameba's ability to bind and lyse target cells and compare it to E. histolytica which has been axenically cultured for years. We found that under axenic conditions, E. dispar's adherence to target cells, ligand binding, and cytotoxicity were less than that of E. histolytica. These events were Gal/GalNAc-inhibitable supporting the idea that E. dispar expresses a lectin similar to E. histolytica. Genetic analysis showed that E. dispar had at least two members of the lectin heavy subunit family and four members of the lectin light subunit family that hybridized to ehhgl and ehlgl gene probes. A library screen produced clones which were isolated and sequenced. Derived amino acid sequences showed that the E. dispar heavy and light subunit clones were 86% and 79% identical, respectively, to their E. histolytica counterparts. In particular, the region which contains the epitopes for two adherence-enhancing monoclonal antibodies and a complement-sensitizing monoclonal antibody (amino acids 882–959 of the lectin heavy subunit) were conserved between the species.     

Oxidative folding and reductive activities of EhPDI, a protein disulfide isomerase from Entamoeba histolytica

PDI enzymes are oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. We have previously identified an E. histolytica PDI enzyme (EhPDI) that exhibits oxidase activity in vivo. However, little is known about the specific role of its redox-related structural features on the enzymatic activity. Here, we have studied the in vivo oxidative folding of EhPDI by mutagenic analysis and functional complementation assays as well as the in vitro oxidative folding and reductive activities by comparative kinetics using functional homologues in standard assays. We have found that the active-site cysteine residues of the functional domains (Trx-domains) are essential for catalysis of disulfide bond formation in polypeptides and proteins, such as the bacterial alkaline phosphatase. Furthermore, we have shown that the recombinant EhPDI enzyme has some typical properties of PDI enzymes: oxidase and reductase activities. These activities were comparable to those observed for other functional equivalents, such as bovine PDI or bacterial thioredoxin, under the same experimental conditions. These findings will be helpful for further studies intended to understand the physiological role of EhPDI.     

Cell-free synthesis and combinatorial selective N-labeling of the cytotoxic protein amoebapore A from Entamoeba histolytica

Amoebapore A is a pore-forming protein produced by the pathogenic parasite Entamoeba histolytica, which causes human amoebic dysentery. The pore-forming activity of amoebapore A is regulated by pH-dependent dimerization, a prerequisite for membrane insertion and pore formation. Understanding of these important processes has been hampered by the cytotoxicity of amoebapore A, which prevents the production of this protein in cell-based expression systems. In this study, a protocol for the cell-free production of active recombinant amoebapore A is presented. Protein yields of 500 μg/ml of cell-free reaction were achieved. Recombinant amoebapore A was purified using a three-step procedure. To facilitate the structural characterization of the dimeric and pore forms, we adapted the cell-free system to isotope label amoebapore A for NMR studies. The preliminary assignment of a 2D ¹H–¹⁵N HSQC spectrum of a uniformly ¹³C/¹⁵N-labeled sample was achieved using a combinatorial selective ¹⁵N-labeling approach coupled with available ¹H_N chemical shift data, resulting in the unambiguous assignment of resonances from 55 of the 77 residues. To confirm these results and obtain the full sequence-specific assignments of the 2D ¹H–¹⁵N HSQC spectrum, a 3D HNCA spectrum was recorded. These assignment data will be used to aid the characterization of amoebapore A dimer formation and membrane insertion.     

Entamoeba histolytica and Trichomonas vaginalis: Trophozoite growth inhibition by metronidazole electro-transferred water

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 Trichomonasvaginalis 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.     

Expression and functional analysis of an Entamoeba histolytica truncated adhesin in tomato plants

Several antigens from Entamoeba histolytica the agent causing human amoebiasis have been identified as potential vaccine candidates. One of them is the EhADH112 adhesin, which plays an important role during trophozoite adherence to and phagocytosis of target cells. We report here the expression of the EhADH112 carboxy-terminus of the gene (Adh240) in tomato plants. A 28 kDa band is recognized by specific antibodies in total proteins from transformed tomatoes. The plant-based adhesin conserves the ability to inhibit trophozoite adherence and phagocytosis of target cells by an average of 32.8 and 44.75% respectively.     

Entamoeba histolytica : gene linkage groups and relevant features of its karyotype

 We identified some gene linkage groups in Entamoeba histolytica using a 4-M urea improved transversal alternating field electrophoresis (TAFE) method. Complex rosette-structured DNA molecules were found trapped along the gel lanes, explaining the fuzziness of the patterns. Using several episomal probes, including 16 S, 5.8 S, and 25 S ribosomal (r)Dna genes, an autonomous replication sequence (ARS), and EhVR1, we identified a complete ribosomal episome linkage group (CELG) at the 1.2-Mb position. Three other incomplete groups were found: IELG-1, formed by EhVR1,16 S, 5.8 S, and 25 S genes; IELG-2 formed by EhVR1, 16 S and 25 S; and IELG-3 formed only by 5.8 S. Ehadh3, Ehpfo, and Ehredox genes migrated at the 1.8-Mb position, forming the non-ribosomal linkage group, NRLG-1.8, while the Ehenl-1 gene migrated at 1.6 Mb forming the NRLG-1.6 group. Ehhk was located at 1.2, 0.8, and 0.17 Mb in three different groups: NRLG-1.2, IELG-3-0.8, and NRLG-0.17. Putative lineal chromosomes were also identified using an heterologous telomeric probe. By in situ hybridization experiments, the rDNA and Ehhk genes were located in both nucleus and cytoplasm, while the Ehpfo and Ehredox genes were found mainly in the nucleus. We propose a model hypothezising that the 16 S and 25 S genes are in a linear molecule, duplicated in two inverted repeats, which may be looped out of the linear DNA to form an episome probably lacking or not the 5.8 S sequence, which could be added later by recombination. Received: 22 March 1996 / Accepted: 11 July 1996     

Serodiagnosis of amoebiasis using a recombinant protein fragment of the 29 kDa surface antigen of Entamoeba histolytica

To develop an improved serodiagnostic test for amoebiasis, we performed a detailed analysis of the immunodominant epitopes of the 29 kDa surface antigen and evaluated its sensitivity and specificity. Enzyme-linked immunosorbent assay (ELISA) based on the fragment containing the immunodominant epitope was evaluated further and compared with full-length recombinant 29 kDa protein. Specificity and sensitivity of the two ELISAs were assessed using 55 human sera of parasitic protozoa infection cases (25 amoebiasis, 20 giardiasis and 10 toxoplasmosis sera) and 10 healthy control sera. The immunodominant epitope of the 29 kDa antigen is localised only in the N-terminus 14–54 amino acid residues. The sensitivities of the two ELISAs were very high, 92 and 96%, respectively. The specificity of the fragment was 100%, whereas the specificity of the full-length 29 kDa protein was 86.6%. These results indicate that the fragment containing the immunodominant epitope of the 29 kDa protein can be used to accurately serodiagnose amoebiasis without cross-reactivity from other parasites.     

Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens

McCaul T.F. and Bird R.G. 1978. Localisation of thiamine pyrophosphatase within the cytoplasmic fine structure of trophozoites of Entamoeba histolytica and E. invadens. International Journal for Parasitology8: 501–506. The distribution of thiamine pyrophosphatase (TPPase) activity was studied in both formaldehyde and glutaraldehyde fixed trophozoites of Entamoeba histolytica and E. invadens. The activity was localised within certain vacuoles. No dense deposits for TPPase activity were seen in the small vesicles, elongated smooth-walled lacunae equated with endoplasmic reticulum, or the nucleus. The demonstration of small vesicles surrounding the larger vacuoles indicated that the Golgi-like vacuoles might be involved in the production of cell coat materials and primary lysosomes.     

Differences in adhesion, phagocytosis and virulence of clones from Entamoeba histolytica, strain HM1: IMSS

Orozco E., Suárez M. E. and Sánchez T. Differences in adhesion, phagocytosis and virulence of clones from Entamoeba histolytica, strain HM1: IMSS. International Journal for Parasitology15: 655–660. Clones isolated from Entamoeba histolytica, strain HM1: IMSS were tested for adhesion, phagocytosis and virulence after subculturing in liquid medium. Other clones were isolated from a subpopulation of strain HM1: IMSS, and highly phagocytic trophozoites were eliminated by irradiation, after incorporating bromodeoxiuridine into their DNA by phagocytosis of labelled bacteria. We thus obtained several clones from strain HM1: IMSS showing a different degree of phagocytosis. Some phagocytosis-deficient clones showed impairment in red blood cell adherence, while others showed a reduced intake of particles into their cytoplasm. The degree of phagocytosis always was associated with the virulence of the clone.     

Isolation of pure compound R/J/3 from Pluchea indica (L.) Less. and its anti-amoebic activities against Entamoeba histolytica

The plant Pluchea indica is known for its anti-inflammatory, anti-ulcer, anti-pyretic, hypoglycemic, diuretic and anti-microbial activities besides many other pharmacological activities. We have isolated and purified seven compounds from the methanolic root extract of this plant by column chromatography. The compounds were identified by spectroscopic analyses. The anti-amoebic activities of the pure compound R/J/3 was investigated against the HM1 strain of Entamoeba histolytica. The compound, R/J/3 showed the most pronounced anti-proliferative activity at a dose of 50 microg/ml. It also showed a marked activity on cell lysis of trophozoites, 4h after administration. The cell lytic activity was compared with metronidazole (5 microg/ml) as positive control.     

Entamoeba invadens: Identification of ADF/cofilin and their expression analysis in relation to encystation and excystation

The differentiation processes of excystation and encystation of Entamoeba are essential for infection and completion of their life-cycle, and the processes need cell motility and its control by actin cytoskeletal reorganization. This study investigated actin depolymerizing factor (ADF)/cofilin (Cfl) family proteins, which are important molecules in actin cytoskeletal reorganization, in Entamoeba invadens in relation to the encystation and excystation. Axenic culture systems were used to induce encystation and excystation. A homology search of the E. invadens genome database and molecular cloning identified three ADF/Cfl family proteins of the parasite (named for short as EiCfl-1, EiCfl-2, and EiCfl-3). This is different from other Entamoeba species, i.e. Entamoeba histolytica and Entamoeba dispar, each of which has only one ADF/Cfl family protein. These ADF/Cfl of E. invadens do not have Ser3 (serine locates third from first methionine), similar to E. histolytica, E. dispar, Saccharomyces cerevisiae and Schizosaccharomyces pombe, although the activity of ADF/Cfl is negatively regulated by phosphorylation of the Ser3 in metazoans. Phylogenetic analysis revealed that Entamoeba Cfl formed a distinctive clade that is separate from other organisms, and the branches of the tree were separated in two consistent with the presence and absence of Ser3. Rabbit anti-EiCfl-2 serum reacted with all recombinant EiCfls and EiCfl in lysates of cysts, trophozoites and metacystic amoebae. Immunofluorescence staining with this antiserum showed co-localization of EiCfl with actin beneath the cell membrane through the life stages. Both proteins proved to be rich in pseudopodia of trophozoites and metacystic amoebae. Real-time RT-PCR showed that mRNAs of EiCfl-2 and actins were highly expressed, but there were few mRNA of EiCfl-1 and EiCfl-3. Remarkably decreased mRNA levels were observed in EiCfl-2 and actins during encystation. All three EiCfls and actins became transcribed after the induction of excystation. The mRNAs of only EiCfl-1 and EiCfl-3 increased remarkably when the excystation was induced in the presence of cytochalasin D. These findings demonstrate that EiCfl-2 and actins co-localize beneath the cell membrane in trophozoites and cysts as well as metacystic amoebae being rich in pseudopodia, that EiCfl-1 and EiCfl-3 are expressed only after the induction of excystation, and that enhanced excystation by cytochalasin D is associated with high expression of EiCfl-1 and EiCfl-3.     

Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.     

Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.     

Short interfering RNA-mediated gene targeting in the zebrafish

Short interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function in plants, invertebrates and mammalian systems. Here we report the use of siRNAs for targeting the zebrafish dystrophin gene. This study demonstrates the efficacy of siRNA-based gene silencing in this vertebrate model species, and illustrates the potential of this approach for determining the roles of multiple protein products expressed by a single gene during the early stages of development. In addition this study illustrates the usefulness of zebrafish as a model for muscle disease, and highlights the potential of siRNA-based gene targeting for disease analysis in this model organism.