A DNA probe specific to pathogenic Entamoeba histolytica.

A DNA sequence, IE-gen1 (3.1 kb), was isolated from the pathogenic strain of E. histolytica NIH-200. IE-gen1 was identified by the subtractive hybridization of a genomic library to a cDNA probe prepared from NIH-200 trophozoites. The IE-gen1 probe specifically detected pathogenic E. histolytica in slot blots of genomic DNA and Northern blots, but not other Entamoeba species and additional human parasites. This genomic probe could detect with complete specificity DNA from about 10(3) organisms. The IE-gen1 probe could be related to highly specialized loci in pathogenic E. histolytica, and is likely to be a valuable DNA reagent for clinical diagnosis and epidemiological investigations.     

Molecular characterization of IgG and IgE antigens of Entamoeba histolytica.

Several surface IgG antigens of E. histolytica have been characterized. We isolated two novel cDNAs by screening a library of pathogenic E. histolytica with rabbit antiserum against a membrane preparation of pathogenic amebas. cDNA K15 encodes a large protein > 220 kDa with sequence homology to myosins and K18 encodes a protein of around 40 kDa. Serum IgG from patients suffering from amebiasis binds to both antigens. Some IgE antibodies are able to recognize homologous proteins in all eukaryotes investigated. We demonstrate that IgE antibodies from pollen allergic patients bind to plant, human, as well as E. histolytica profilins.     

Macrophage colony-stimulating factor enhances the respiratory burst of human monocytes in response to Entamoeba histolytica.

Mononuclear phagocytes (MP) are probably the most capable effector cells of the body in the defense against virulent strains of E. histolytica. Killing of E. histolytica by MP appears to involve both oxidative and non-oxidative mechanisms. Thus, in this study we have investigated whether trophozoites of an axenic virulent strain E. histolytica HM1:IMSS (EH) were capable of eliciting an oxidative response in pure populations of freshly isolated human monocytes. Using a luminol-enhanced chemiluminescence assay we demonstrate that these cells produce a strong respiratory burst when challenged with live amebas over a wide range of MP:EH ratios. Furthermore, pre-incubation of monocytes with recombinant Macrophage Colony Stimulating Factor (M-CSF) could further increase the oxidative metabolism of MP in response to E. histolytica. Our results indicate that, in contrast to what occurs with polymorphonuclear leukocytes, the interaction of E. histolytica with MP leads to the production of reactive oxygen intermediates by this cells. The enhancement of this potent microbicidal mechanism by inflammatory cytokines may further increase the amebicidal capacity of human mononuclear phagocytes.     

Interaction of various Entamoeba histolytica strains with human intestinal cell lines.

The interaction of four pathogenic and three nonpathogenic E. histolytica strains with two human intestinal cell lines (Caco-2 and HT-29) was examined. The adherence of pathogenic and nonpathogenic E. histolytica to these cells was similar, indicating that defective adherence to intestinal cells is not a common feature of nonpathogenic strains. The addition of different carbohydrates confirmed the importance of the galactose-binding lectin of E. histolytica in binding to these intestinal cells. On the other hand, only virulent E. histolytica strains damaged monolayers of intestinal cells. The results indicate that Caco-2 cells and differentiated HT-29 cells are useful models for research of intestinal amebiasis.     

Host tissue invasion by Entamoeba histolytica is powered by motility and phagocytosis

During amebiasis, E. histolytica motility is a key factor to achieve its progression across tissues. The pathogenicity of E. histolytica includes its capacity to phagocyte human cells. Motility requires polarization of E. histolytica that involves protrusion of a pseudopod containing actin and associated proteins [myosin IB, ABP-120 and a p21-activated kinase (PAK)] and whole-cell propulsion after contraction of the rear of the cell, where myosin II and F-actin are concentrated. An interesting characteristic of this parasite is the presence of two unique myosins (myosin II and unconventional myosin IB), in contrast to several actin genes. Little is known about the regulation of the actin-myosin cytoskeleton dynamics of E. histolytica, and a better understanding of signaling pathways that stimulate and coordinate regulators protein and cytoskeleton elements will provide new insight into the cell biology of the parasite and in amebiasis. Here we summarize the pleiotropic functions described for myosin II and PAK in E. histolytica. We propose that survival and pathogenicity of E. histolytica require an active actin-myosin cytoskeleton to cap surface receptors, to adhere to host components, to migrate through tissues, to phagocyte human cells and to form liver abscesses.     

PCR法检测粪便样本溶组织内阿米巴的初步研究

  目的  建立直接检测粪便样本中溶组织内阿米巴的PCR方法。  方法  根据溶组织内阿米巴标准株基因组中小亚基核糖体RNA（small subunit ribosome RNA,SSU rRNA）序列合成4对特异性引物（2对自主设计引物和2对参考引物）,建立PCR检测方法,用该方法对221例临床腹泻患者粪便标本进行检测,同时采用病原学碘染涂片镜检法和酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测抗原,对3种方法的阳性率进行比较,并采用Kappa检验进行一致性分析,分析3种检测方法结果的准确性。  结果  4对引物均扩增出溶组织内阿米巴特异的目的片段,建立了粪便样本溶组织内阿米巴检测的PCR法。选择其中2对引物（1对自主设计引物和1对参考引物）对221例粪便样本进行PCR扩增,同时用碘染涂片镜检法查病原体,用ELISA法进行抗原检测,3种方法溶组织内阿米巴阳性检出率分别为2.26%、0.90%和9.50%,差异有统计学意义（χ2=23.34, P<0.01）。PCR法与碘染涂片镜检法比较,Kappa值为0.216,一致性微弱;PCR法与ELISA法比较,一致性差,Kappa值为–0.134。PCR法的结果与临床诊断的一致性最好。  结论  本研究通过自行设计引物建立的粪便样本溶组织内阿米巴PCR检测法在准确性上优于镜检法和ELISA抗原检测法,为该方法用于临床诊断提供了实验基础。     

The fast release of mucin secretion from human colonic cells induced by Entamoeba histolytica is dependent on contact and protein kinase C activation.

The mucus producing colonic cell line, LS174T, was used as a model to study E. histolytica-induced mucin secretion. E. histolytica trophozoites in contact with the mucus layer overlying the LS174T cells and in response to PMA, a protein kinase C activator, and Ca2+ ionophore A23187 which elevates intracellular Ca2+ ([Ca]i), caused a time-dependent (0.25-2.00 h) release of mucin. PKC inhibitors, H7 and staurosporine inhibited E. histolytica (37 and 75%) and PMA (46 and 100%)-induced mucin secretion, whereas in response to Ca2+ ionophore mucin secretion was augmented (56 and 17%). Both PMA and E. histolytica-induced the translocation of the PKC enzyme from the cytoplasm to the membrane fraction with increased enzyme activity. These results suggest that even though mucin secretion can be induced by PKC and Ca(2+)-dependent pathways, E. histolytica evokes the fast release of mucins by a PKC-dependent mechanism.     

Partial nucleotide sequence of the enzyme pyruvate, orthophosphate dikinase of Entamoeba histolytica HM1:IMSS.

We isolated a clone from a lambda gt11 cDNA library of E. histolytica, subcloned its insert in the vector pTZ18R and sequenced it by the method of Sanger. Sequence comparison with Genbank and of the corresponding amino acid sequence with the NBRF/PIR data bank using the FastA program, showed homologies between 54.9 and 58.5% for the nucleotides and between 48.6 and 49.4% for the amino acids with pyruvate, orthophosphate dikinases from maize, Flaveria trinervia and Bacteroides symbiosus. The sequence obtained for E. histolytica represents about 20% of the complete gene or protein sequence.     

Methods for the investigation of diversity in Entamoeba histolytica

The ability to distinguish variants of a species has many potential applications. In Entamoeba histolytica the first method to detect variation was based on isoenzyme analysis. However, this approach has been superseded by DNA-based analysis. In this review I discuss the basis of the variation detected in E. histolytica by the various molecular methods that have been published to date. Information on diversity in other species is mentioned where such information exists.     

Actin associated proteins of Entamoeba histolytica.

Treatment of E. histolytica HM1-IMSS trophozoite extracts to conditions that produce gels of actin and associated cytoskeletal proteins in other ameboid cells caused formation of macroscopic actin rich complexes (ARCs). The one-dimensional PAGE protein profile of this ARC was similar to those of Dictyostelium and Acanthamoeba actin gels. Formation of the E. histolytica ARCs was enhanced by added lipids. In addition to actin, the ARC was enriched with proteins that showed cross-reactivity to antibodies to alpha-actinin and the 50K actin binding protein (elongation factor 1 alpha) from Dictyostelium. E. histolytica ARCs appear to be comprised of a number of actin cytoskeleton proteins and provide a source for their isolation and characterization.     

A survey of Entamoeba histolytica and Entamoeba dispar (Brumpt) infections on Mahé, the Seychelles.

E. histolytica, pathogenic, and E. dispar, non-pathogenic, being morphologically identical are, when recovered as cysts in feces, diagnosed as E. histolytica. A survey on The Seychelles demonstrated that when culture and zymodeme characterization was used to compare against microscopy alone, the risk of overdiagnosis of E. histolytica infections was drastically reduced. From a total of 313 subjects tested 21 cultures grew amebas. By zymodeme analysis eight were E. histolytica, 40 were E. dispar and the remainder were various species of non-pathogenic intestinal amebas. Two further small comparative surveys confirmed these findings.     

The fine structure of Entamoeba histolytica processed by cryo-fixation and cryo-substitution.

Recently, the use of cryo-fixation followed by freeze-substitution has been shown to produce a better ultrastructural preservation of cellular components due to the rapidity of the fixation procedure and the lack of distortion during dehydration. When these techniques are applied to study the fine structure of axenically cultured trophozoites of E. histolytica, an improved preservation of cytoplasmic and nuclear components was found. The surface coat of the parasite appears much thicker and uniform than in conventionally treated samples. Also, the ground substance of the cytoplasm is packed with fibrogranular material, which is usually extracted by chemical fixation. The extremely fast fixation procedure allows the visualization of fusion and/or fission processes of cytoplasmic vacuoles and vesicles. Nuclear microtubules and cytoplasmic microfilaments are clearly identified. Low-temperature techniques allow not only a better preservation of the cell structure of E. histolytica parasites, but will also facilitate considerably future immunoelectronmicroscopical studies.     

Characterization of two long vesicle-associated membrane proteins or longins genes from Entamoeba histolytica

BACKGROUND: This study characterizes two long vesicle-associated membrane protein (VAMP) genes (SYBL-1 and SYBL-2) obtained from DNA of Entamoeba histolytica by PCR amplification. (Nucleotide sequences of the Entamoeba histolytica SYBL-1 and SYBL-2 genes appear in the GeneBank under accession numbers AY256852 and AY309014.) METHODS: The two cDNA products include one of 663 bp with sequence of 220 deduced amino acids, and a second of 693 bp with 230 deduced amino acid residues. Both products possess corresponding sequences for longin domain at N-terminus, a soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor (SNARE) coiled-coil region, a transmembrane domain (TM), and an intravesicular tail C-terminal, characteristics of all long VAMPs or longins. The current study identified presence of deduced peptide bonds in SYBL-1 and -2, which indicates that these two long VAMPs from E. histolytica could be appropriate substrates for zinc endopeptidases from tetanus and botulinum neurotoxins with specific activity toward neuronal synaptobrevins. RESULTS: Alignment by Basic Local Alignment Search Tool (BLAST) of deduced amino-acid sequence of long VAMPs genes SYBL-1 and -2 showed identity of between 20 and 40% with Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus, Homo sapiens, and Plasmodium falciparum. CONCLUSIONS: It is possible that these two putative transport proteins are involved in endocytosis/exocytosis during a biological membrane fusion process, which may make them suitable candidates as targets for new drugs. According to published data, this is the first time that two such genes have been isolated from E. histolytica.     

溶组织阿米巴肠炎诊断及药物治疗研究进展

阿米巴（Amoeba）词源自希腊文amoibē,属原生动物变形虫科,当该虫移动或摄取营养时,胞浆可伸出伪足而改变其外形,故而得名“变形虫”。阿米巴在地球上存在了几十亿年,拥有极强的适应能力,可随外界环境变化不断地自我调整。目前,有4种形态完全相同的内阿米巴可对人类造成感染,即溶组织内阿米巴、迪斯帕内阿米巴（E. dispar）、莫氏内阿米巴（E. moshkovskii）和孟加拉阿米巴（E. bangladeshi）。其中,溶组织内阿米巴是明确可导致人类患病的病原体,且人群对溶组织内阿米巴普遍易感,感染后对该病无免疫力。肠道作为溶组织内阿米巴滋养体首先定居和侵袭的部位,绝大多数患者均以肠道症状而就诊。近年来,溶组织内阿米巴肠炎诊断的敏感性和特异性得到了极大提高,但其治疗的有效药物相对较少。本文通过综述近10年来国内外文献中溶组织内阿米巴诊断、治疗的最新进展,对提高国内临床医师对溶组织内阿米巴肠炎诊断和治疗的认识有指导意义。     

Electron microscopic enzyme cytochemistry of Entamoeba histolytica trophozoite]

Electron microscopic enzyme cytochemical reactions of Entamoeba histolytica trophozoite showed that acid phosphatase (ACP) and cytidine monophosphatase (CMPase) were located in the lysosomes. The lysosome containing enzymes were distributed in the endoplasm and beneath the plasmalemma, and the releasing enzymes by lysosomes excreted outside of the plasmalemma and caused the injury to host cells. The cytochemical positive reactions of catalase and glucose-6-phosphatase (G-6-Pase) showed that E. histolytica contains microbodies and endoplasmic reticulum. The reactive products of peroxidase (POase) were seen in the lysosome-like structure. The reactions of cytochrome oxidase (COase) and succinate dehydrogenase (SDH) were both negative, indicating that E. histolytica lacked mitochondria. The reactions of thiamine pyrophosphatase (TPPase) and nicotinamide adenine dinucleotide phosphatase (NADPase) were both negative, indicating that E. histolytica lacked Golgi body. The reactions of Na(+)-K(+)-ATPase were located on plasmalemma.     

Calcium-binding proteins of Entamoeba histolytica

Calcium plays an essential role in many fundamental processes in almost all eukaryotic cells including protozoan parasite Entamoeba histolytica. Many of the calcium-mediated processes are carried out through the help of calcium-binding proteins (CaBPs). A few of these E. histolytica CaBPs have been described before. These proteins are unique to this organism and are thought to be essential. Availability of genome sequence has opened up the possibility of studying CaBPs at the whole genome level. In this preliminary report, we describe the complement of CaBPs present in E. histolytica. A large fraction of these genes are expressed in the trophozoites and are likely to be functional. The results suggest a number of pathways that are involved in calcium signaling and may be unique for this organism.     

Biochemical analysis of Entamoeba histolytica HM1 strain resistant to complement lysis.

In this study, quantitation of several hydrolases and analysis of electrophoretic patterns of E. histolytica HM1 axenic strain periodically exposed to lytic-human serum (lytic-HS), heat-inactivated human (HI-HS) or bovine (HI-BS) sera were performed. The N-acetylglucosaminidase (NAGAse) levels increased in amebas resistant to complement. The acid phosphatase (AP) and proteolytic activity (PA) levels did not vary significantly in all groups of treated amebas, as compared with amebas from axenic cultures. Moreover, amebas exposed to either HI-HS or lytic-HS sera showed a discrete quantitative increase as compared with HI-BS group, in some peptides with a M(r) between 116 and 45 kDa. These results indicate that, besides resistance to complement lysis, exposure of E. histolytica to lytic-HS induces an augmentation in NAGAse levels.     

Primary structures of alcohol and aldehyde dehydrogenase genes of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite E. histolytica under the anaerobic conditions found in the lumen of the colon. With the goal of finding new targets for anti-amebic drugs, the E. histolytica NADP(+)-dependent alcohol dehydrogenase gene (EhADH1; EC 1.1.1.2) and an aldehyde dehydrogenase gene (EhALDH1; EC 1.3.2) were cloned. The EhADH1 alcohol dehydrogenase gene encoded -39 kDa protein with 62 and 60% amino acid identities, respectively, with NADP(+)-dependent alcohol dehydrogenases of anaerobic bacteria Thermoanaerobium brockii and Clostridia beijerinckii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. An EhADH1-glutathione-S-transferase fusion protein showed the expected NADP(+)-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADH1 fusion protein were inhibited by pyrazole and 4-methyl pyrazole. The E. histolytica aldehyde dehydrogenase EhALDH1 gene encoded a 60 kDa protein, which showed a 36% amino acid identity over a 451 amino acid overlap with the human stomach aldehyde dehydrogenase (ALDH3).     

溶组织内阿米巴ELISA试剂盒在阿米巴病调查的应用

目的了解景洪市爱尼族人群阿米巴病流行情况,为阿米巴病监测和防治提供依据,并评价ELISA试剂盒在阿米巴病调查应用的效果。方法以整群随机抽样爱尼族村寨,逐户收集新鲜粪便标本,用观察法、碘液涂片镜检法和TECHLAB第二代溶组织内阿米巴ELISA试剂盒对照检测粪便标本溶内阿米巴。结果镜检278例,E·h/E·d感染为9·71%;ELISA检测366例,E·h感染率为9·29%;镜检和ELISA共同阳性15例,占镜检阳性的55·56%;粪便观察和ELISA检测结果诊断5例为肠阿米巴病,发病率为1·37%,E·h致病率为14·71%。结论景洪爱尼族阿米巴病流行严重,应加强阿米巴病的监测和防治工作,同时积极推广新技术。