Transient transfection and expression of firefly luciferase in Giardia lamblia.

We have developed a gene transfer system for the protozoan parasite Giardia lamblia. This organism is responsible for many cases of diarrhea worldwide and is considered to be one of the most primitive eukaryotes. Expression of a heterologous gene was detected in this parasite after electroporation with appropriate DNA constructs. We constructed a series of transfection plasmids using flanking sequences of the Giardia glutamate dehydrogenase (GDH) gene to drive expression of the firefly luciferase reporter gene. The optimal construct consisted of a GDH/luciferase fusion gene in which the first 18 codons of the GDH gene immediately preceded the luciferase gene; this fusion gene was flanked by the upstream and downstream sequences of the GDH gene. Electroporation of this construct into Giardia yielded luciferase activity that was 3000- to 50,000-fold above background. Removal of either the 5' or 3' GDH flanking sequences from this construct resulted in significantly reduced luciferase activity, and removal of both flanking sequences reduced luciferase activity to background levels. Luciferase activity was proportional to the amount of DNA electroporated and was maximal at 6 hr after electroporation.     

Transfection of the malaria parasite and expression of firefly luciferase.

The goal of this work is to develop a method for the functional analysis of malaria genes using the method of DNA transfection. We have developed a transient transfection vector by constructing a chimeric gene in which the firefly luciferase gene was inserted in frame into the coding region of the pgs28 gene of Plasmodium gallinaceum. This plasmid DNA was introduced into P. gallinaceum gametes and fertilized zygotes by electroporation, and luciferase expression was assayed after 24 hr. This report of successful introduction and expression of a foreign gene in a malaria parasite demonstrates the feasibility of this approach to developing methods for the functional analysis of parasite genes.     

Transfection and continuous expression of heterologous genes in the protozoan parasite Entamoeba histolytica.

To provide tools for functional molecular genetics of the protozoan parasite Entamoeba histolytica, we investigated the use of the prokaryotic neomycin phosphotransferase (NEO) gene as a selectable marker for the transfection of the parasite. An Escherichia coli-derived plasmid vector was constructed (pA5'A3'NEO) containing the NEO coding region flanked by untranslated 5' and 3' sequences of an Ent. histolytica actin gene. Preceding experiments had revealed that amoebae are highly sensitive to the neomycin analogue G418 and do not survive in the presence of as little as 2 micrograms/ml. Transfection of circular pA5'A3'NEO via electroporation resulted in Ent. histolytica trophozoites resistant to G418 up to 100 micrograms/ml. DNA and RNA analyses of resistant cells indicated that (i) the transfected DNA was not integrated into the amoeba genome but was segregated episomally, (ii) in the amoebae, the plasmid replicated autonomously, (iii) the copy number of the plasmid and the expression of NEO-specific RNA were proportional to the amount of G418 used for selection, and (iv) under continuous selection, the plasmid was propagated over an observation period of 6 months. Moreover, the plasmid could be recloned into E. coli and was found to be unrearranged. To investigate the use of pA5'A3'NEO to coexpress other genes in Ent. histolytica, a second marker, the prokaryotic chloramphenicol acetyltransferase (CAT) gene under control of an Ent. histolytica lectin gene promoter was introduced into the plasmid. Transfection of the amoebae with this construct also conferred G418 resistance and, in addition, allowed continuous expression of CAT activity in quantities corresponding to the amount of G418 used for selection. When selection was discontinued, transfected plasmids were lost as indicated by an exponential decline of CAT activity in trophozoite extracts.     

Transfection and transient expression of chloramphenicol acetyltransferase gene in the protozoan parasite Entamoeba histolytica.

Hybrid plasmids were constructed and used for successful transfection and transient expression of the chloramphenicol acetyltransferase (CAT) gene in the protozoan parasite Entamoeba histolytica. Transfection was performed by electroporation of the amebae in a potassium phosphate-based buffer under conditions of 3000 V/cm and 25 microF, resulting in a time constant of 0.4 ms. Expression of CAT activity was achieved with constructs in which the CAT coding region was flanked by untranslated upstream and downstream sequences of E. histolytica genes. Highest activity was detected after culturing transfected cells for 48 hr. Activity was found to be proportional to the amount of DNA transfected.     

Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.

A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.     

阳离子脂质体介导的虫荧光素酶基因表达

目的 研究阳离子脂质体Lipofectin介导虫荧光素酶基因在不同细胞株中的表达。方法 质粒pDR2luc在大肠杆菌DH5a中扩增后用碱裂解法提取,并经Sepharose 2B凝胶过滤柱层析,通过凝胶电泳及紫外分光光度计分析纯度、定量,以Lipofectin分别转染人肝癌细胞株HepG2、SMMC7721、人肾癌细胞株GRC及非洲绿猴肾细胞COS7,然后以液体闪烁计数仪单光子计数法测定荧光素酶活性。结果 荧光素酶活性在4种细胞株中均有表达,且均有显著性差异(均P<0.05)。结论 阳离子脂质体Lipofectin可用于多种真核细胞基因转染。     

The rhabdoviruses of Entamoeba histolytica and Entamoeba invadens.

Rhabdoviruses have been described in plants, arthropods and vertebrates including man. Members of the group are of agricultural, veterinary and medical importance. The presence of a rhabdovirus in Entamoeba histolytica and Entamoeba invadens is the first record of their existence within protozoa. The morphology of this virus is described and its significance discussed, in relation to a possible lysogenic state and pathogenecity of Entamoeba species.     

Pore-forming peptide of pathogenic Entamoeba histolytica.

A polypeptide that causes pore formation in target-cell membranes is implicated in the potent cytolytic activity of pathogenic Entamoeba histolytica. Pore-forming material was purified to apparent homogeneity by a multistep procedure, and its analysis by NaDodSO4/PAGE revealed one peptide of 4-5 kDa under nonreducing or under reducing conditions. Pore-forming activity was measured by depolarization of liposome membrane potential and was found to be optimally expressed at low pH. Active material preferentially inserted into negatively charged lipid vesicles. Treatment of purified amoeba peptide in solution or bound to liposomes with glutaraldehyde revealed oligomers upon NaDodSO4/PAGE, suggesting functionally relevant peptide-peptide interactions. The NH2-terminal amino acid sequence of the amoeba peptide was determined by protein sequencing and revealed a structural similarity to melittin, the membranolytic peptide of bee venom.     

Entamoeba histolytica infection in male homosexuals.

Amoebic infection in two male homosexuals is described. The possibility that this infection was acquired through homosexual practices and the implications to clinical and diagnostic services is discussed.     

Antigenicity of sub-cellular components of Entamoeba histolytica.

Antigencity of the sub-cellular components of axenic Entamoeba histolytica trophozoites was studied. The cells were disrupted by means of a glass-teflon homogeniser and sub-cellular components were prepared by stepwise differential centrifugation. Four fractions were obtained, namely the 350 g, 6500 g, and 100,500 g fractions and the cell sap. Components of the sedimented fractions were examined by phase contrast and electron microscopy. The antigenicity of each fraction was studied by two different methods:-(1) By extraction with 0.5% sodium deoxycholate followed by testing against the reference sera; (2) By demonstration of the loss of immunological activity of the reference sera after absorption with fractionated components. It was found that all 4 fractions had varying antigenic activities as measured in the indirect hemagglutination (IHA), the complement fixation (CF) and the immunoelectrophoresis (IEP) tests. With the extraction technique, the following results were obtained:- The highest IHA activity was found in the cell sap, whereas this activity in other fractions clustered at lower levels. In the CF test, the activities associated with all 4 fractions were similar. In the IEP test, the highest activity was found in the cell sap and the least activity in the 100,500 g fraction. With the absorption technique, slightly different results were obtained. Whilst in the IHA and the IEP test, the results were in concordance with the extraction technique, the CF activity was slightly different, since it was highest in the cell sap and least in the 100,500 g fraction.     

Cloning and expression of an NADP(+)-dependent alcohol dehydrogenase gene of Entamoeba histolytica.

Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entamoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoelectric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP(+)-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADH1 clone had an open reading frame that was 360 amino acids long and encoded a protein of approximately 39 kDa (calculated size). EhADH1 showed a 62% amino acid identity with the tetrameric NADP(+)-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADH1 showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADH1 contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADH1 lacked a 23-amino acid stretch present in other alcohol dehydrogenases that includes four cysteines that bind a second noncatalytic zinc ion. 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-methylpyrazole.     

Susceptibility testing of Entamoeba histolytica

The growth of Entamoeba histolytica in microtiter plates in vitro in a variety of environments with reduced oxygen tensions is reported. With 3% O2, 3% CO2, and 94% N2, the parasite growth in microtiter plates was identical to that in screw-capped culture tubes, as measured by [3H]thymidine incorporation and by quantitative parasite counts. There were no significant differences between the drug concentrations necessary to inhibit parasite growth by 50% based on [3H]thymidine incorporation vs those defined by quantitative parasite counts for the 15 antimicrobial agents tested (including seven drugs used for the treatment of amebiasis). This technique provides a reproducible method to quantitate the activity of potential antiamebic agents in vitro. The isotopic method should be of particular value in defining the metabolism of the parasite and effects of antimicrobial agents on it, whereas the morphologic method may be more valuable for workers with limited resources available to them.     

Cloning and expression of a membrane antigen of Entamoeba histolytica possessing multiple tandem repeats.

Entamoeba histolytica causes amebic dysentery and amebic liver abscess, major causes of morbidity and mortality worldwide. We have used differential hybridization screening to isolate an E. histolytica-specific cDNA clone. The cDNA was found to encode a serine-rich E. histolytica protein (SREHP) containing multiple tandem repeats. The structural motif of SREHP resembles some of the repetitive antigens of malarial species, especially the circumsporozoite proteins. A recombinant trpE fusion protein containing the tandem repeats of SREHP was recognized by immune serum from a patient with amebiasis, demonstrating that SREHP is a naturally immunogenic protein. An antiserum raised against the recombinant fusion protein specifically bound to two distinct bands with apparent molecular masses of 46 and 52 kDa in a crude preparation of E. histolytica trophozoite membranes. This antiserum also inhibited E. histolytica trophozoite adhesion to Chinese hamster ovary cells in vitro. The ability to isolate E. histolytica-specific genes, and to express those genes in Escherichia coli, may be important in studying the molecular basis of E. histolytica pathogenesis and for the future development of vaccines.     

Riboflavin requirement for the cultivation of axenic Entamoeba histolytica.

Riboflavin was found to be essential for the cultivation of axenic Entamoeba histolytica. This is the first demonstration of a flavin requirement by the organism. Panmede, the principal source of flavins in the axenic medium, was treated with activated carbon to remove flavins. Medium made with this flavin-deficient Panmede, and supplemented with ribonucleic acid failed to support the multiplication of amebae in serial subculture, but did so when riboflavin was added. The concentration of riboflavin required to achieve maximal growth was about 1.3 microgram per ml medium. Studies on riboflavin uptake revealed that amebae lack a high-affinity transport system for this vitamin. The rate of riboflavin uptake was equivalent to the rate of pinocytotic uptake of fluid as previously determined.