Molecular and Biochemical Parasitology 99 (1999) 237 – 245
Pantropic retroviral vectors mediate gene transfer and expression in Entamoeba histolytica Xuchu Que a, Doojin Kim a, Alejandro Alagon b, Ken Hirata a, Hiroko Shike c, Chisato Shimizu c, Antonio Gonzalez d, Jane C. Burns c, Sharon L. Reed a,* a
Department of Pathology, Di6ision of Infectious Diseases, Uni6ersity of California, San Diego Medical Center, 214 Dickinson St., San Diego, CA 92103, USA b Instituto de Biotecnologia, Cuerna6aca, Mexico c Department of Pediatrics, Uni6ersity of California, San Diego, CA 92103, USA d Instituto de Parasitologia y Biomedicina, CSIC, Granada, Spain Received 28 September 1998; received in revised form 18 January 1999; accepted 25 January 1999
Abstract Transformation of Entamoeba histolytica has been previously reported, but the foreign genes have all been replicated episomally. Pantropic retroviral vectors based on the Moloney murine leukemia virus with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) have an extremely broad host range and can be concentrated to high titer. To investigate whether these pseudotyped, pantropic vectors can mediate gene transfer and expression in E. histolytica, we constructed a retroviral vector, in which a hygromycin phosphotransferase is expressed from the E. histolytica actin promoter. Data confirm the infection, integration, and expression of a foreign gene mediated by the provirus. To our knowledge, this is the most evolutionarily distant example of successful integration and expression of a mammalian retrovirus. Pantropic retroviral vectors may thus facilitate genetic analysis in species lacking transformation systems. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Entamoeba; Amebiasis; Retroviral vectors; Transformation
Abbre6iations: MoMLV, Moloney murine leukemia virus; VSV-G, envelope glycoprotein of vesicular stomatitis virus; LTR, long terminal repeat. Note: Nucleotide sequences data reported in this paper are available in the EMBL, GenBank™ and DDJB databases under the accession numbers AF113903–AF113907. * Corresponding author. Tel.: + 1-619-5436146; fax: + 1-619-5436614. E-mail address:
[email protected] (S.L. Reed) 0166-6851/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 9 9 ) 0 0 0 2 1 - 3
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1. Introduction Entamoeba histolytica is a protozoan parasite that causes amebic dysentery and liver abscesses. Amebiasis is a common parasitic disease worldwide, surpassed only by malaria and schistosomiasis as a cause of death [1]. Understanding the pathogenesis of invasive amebiasis would be greatly facilitated by the ability to manipulate the genome of E. histolytica, including the ability to introduce and express specific gene products as well as generate insertional mutations. Transfection of DNA sequences into E. histolytica has proven to be difficult, and current methods for the introduction of heterologous DNA sequences into E. histolytica include transient transfection of plasmid DNA and episomally replicating vectors [2 – 7]. The lack of integration has precluded the application of these techniques in gene knock-out and insertional mutagenesis studies. Retroviral vectors, in which foreign DNA sequences are expressed from the viral long terminal repeat (LTR) or internal promoters, are valuable tools for heterologous gene expression in a number of vertebrate species. The range of host cells that can be infected by such vectors is limited to cells expressing a specific protein receptor that mediates the attachment of the viral particles to the cell surface. To overcome this limitation, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus (VSV-G), which binds to phospholipid components in the cell membrane [8]. These pantropic retroviral vectors have an extremely broad host cell range and can infect many non-mammalian species, including fish, marine invertebrates, newt, Xenopus, and insect cell lines [9 – 14]. These vectors can contain up to 10 – 13 kb of heterologous DNA and can integrate stably into the genome of dividing cells. Pantropic retroviral particles can also be concentrated by ultracentrifugation to titers \109 colony-forming units ml − 1 [9,15]. To develop such a gene transfer system for E. histolytica, we tested whether pantropic pseudotyped retroviral vectors could
infect, integrate, and express foreign genes in E. histolytica trophozoites.
2. Materials and methods
2.1. Retro6iral 6ector construction To construct a retroviral vector for E. histolytica, the vector pLNPOZL [16] was digested with XhoI and ClaI to remove the polio ribosomal entry site and the b-galactosidase gene (Lac Z). The XhoI site was repaired with Klenow, and the resulting fragment was ligated with the SacI (trimmed)–ClaI fragment of pBS5%act-hyg-3%L21 containing the ameba actin promoter [17]. This plasmid, pLNAmActHL, contains the Moloney leukemia virus (MoMLV) LTR controlling expression of neomycin phosphotransferase, and the actin promoter of E. histolytica controlling expression of hygromycin phosphotransferase from E. coli, followed by the 3% end of E. histolytica ribosomal protein L21 for expression [18].
2.2. Pseudotyped 6irus production The PA317 and 293 gag–pol packaging cell lines were used for virus production as previously described [15]. Briefly, pLNAmActHL was transfected by standard calcium phosphate co-precipitation into PA317, which expresses high levels of the MoMLV gag, pol, and en6 gene products. Culture supernatants containing amphotropic virus were harvested at 48-h post-transfection and used to infect the 293 gag–pol cell line, which produces high levels of gag and pol gene products, but no envelope protein [9]. Stably transduced colonies were selected in G418. To produce pseudotyped virus, pHCMV-VSV-G (VSV-G expressed from the human cytomegalovirus promoter) was introduced by calcium phosphate co-precipitation, and virus-containing supernatant was harvested at 48–96 h post-transfection, filtered (0.45-mm filter), and stored at −70°C. Virus stocks for infection of E. histolytica trophozoites were concentrated by ultracentrifugation and contained between 1 and 9× 108 colony-forming units (cfu) ml − 1.
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2.3. Infection protocol E. histolytica strain HM1:IMSS trophozoites were maintained axenically in TYI-S-33 medium containing penicillin (100 units ml − 1) and streptomycin (100 mg ml − 1) at 37°C [19]. Trophozoites were grown to logarithmic stage in 75-cm2 plastic flasks for infection experiments. Trophozoites (1 × 106 cells) were resuspended in Optimem/Lcysteine buffer (Gibco-BRL, Gaithersburg, MD) and allowed to attach for 5 min at room temperature to a filter membrane (Costar Transwell tissue culture inserts, Cambridge, MA). The polycation, polybrene (10 mg ml − 1), and 5× 106 cfu of pseudotyped virus (LNAmActHL) in Dulbecco’s modified Eagle’s medium (DMEM) were subsequently added (multiplicity of infection= 5), and allowed to flow past trophozoites for a total contact time of 10 min. The flow-through system has been shown to increase the infection efficiency of retroviral vectors in other settings [20]. For all experiments, negative controls included both trophozoites incubated with non-infectious viral supernatants, in which the particles were produced without the VSV-G envelope glycoprotein and were unable to infect cells (mock-infected control), or with media alone. Following the flowthrough infection, trophozoites were washed twice in TYI-S-33 medium and incubated for 2 h in media alone at 37°C. Hygromycin was then added to the infected and uninfected amebae at a final concentration 7.5 mg ml − 1. To estimate the infection efficiency, hygromycin-resistant trophozoites were counted 4 days post-infection, approximately 48 h after all viable amebae had disappeared from the uninfected control cultures.
2.4. Clonal growth on soft agar Individual ameba colonies were isolated from soft agar culture using a modification of previous procedures [21,22]. Briefly, E. histolytica trophozoites (103 cells) were added to TYI-S-33 soft agar medium containing 8 mg ml − 1 of hygromycin and a final agar concentration of 0.4 – 0.7%. Cloning efficiency was also increased by the addition of 0.05% thioglycollate (BBL). Prior to pouring the soft agar, an extra layer of 1.5% agar was added
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to Petri dishes to inhibit trophozoite motility. The soft agar and amebae mixture was then poured into Petri dishes, chilled and grown anaerobically (Gaspak, BBL) at 37°C. The colonies were visible after 4 days, and individual colonies were inspected microscopically and transferred from agar culture to liquid ameba medium containing 7.5 mg ml − 1 of hygromycin in glass culture tubes. DNA was extracted from the clones for inverse PCR and Southern blot analysis as described below.
2.5. DNA extraction and PCR amplification Trophozoites infected with retroviral vector and controls were grown to confluence under the selection of 7.5 mg ml − 1 hygromycin, and genomic DNA was extracted from retroviral and mock-infected trophozoites using the DEPC-Triton X-100 method [23], combined with the Promega Wizard genomic DNA purification kit. The DNA was further purified with phenol:chloroform extraction, isopropanol precipitation, resuspended in TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA), and the concentration determined by spectrophotometry at 260 nm. Provirus was detected by previously published PCR amplification with primers targeted to the MoMLV LTR [24], which yielded a 244-bp product from the amplification of pLNAmActHL. Conditions for provirus amplification were as follows: 0.5 mM each LTR primer, 1.5 mM MgCl2, 10 mM Tris, pH 8.4, 50 mM KCl, 0.2 mM of each dNTP, and 2.5 U Taq polymerase (Perkin-Elmer, Norwalk, CT) in a reaction volume of 25 ml. The thermocycling parameters were: 95°C×5 min, followed by 40 cycles of 95°C× 1 min, 60°C× 1 min, 72°C× 1 min, and a final extension at 72°C for 5 min. Samples were kept at 4°C until analysis by electrophoresis on a 1.5% agarose gel or transfer to a nylon membrane for hybridization to an LTR probe.
2.6. Amplification of flanking sequences To identify the proviral integration sites, inverse PCR was performed. DNA (5 mg) from individual clones of hygromycin-resistant amebae
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were digested with ScaI and NaeI, extracted in phenol:chloroform, ethanol precipitated, and resuspended in T4 DNA ligation buffer to a concentration of 5 ng ml − 1 to favor intramolecular ligation of the DNA. Reaction mixtures were incubated at 14°C overnight. DNA from the ligation reaction mixture was purified by phenol extraction and ethanol precipitation and resuspended in TE buffer at a concentration of 100 ng ml − 1. Two primers, LTRc 3 (5%-TTATGTATTTTTCCATGCCTTG-3%) and NeoF610 (5%TCTGGATTCATCGACTGTGG-3%), oriented in opposite directions were used for inverse PCR with 200 ng of ligated DNA as template under conditions detailed above. The PCR products were cloned into the pCRII vector (Invitrogen, San Diego, CA) and sequenced (Sequenase 2.0 kit, USB) to determine the integration sites. To confirm the integration of the proviral sequences in amebic DNA, the PCR fragments were labeled by the random-priming method and hybridized to uninfected trophozoite DNA. Sequences were compared to Genbank-deposited sequences using BLASTN.
system in which viral supernatant was simply added to the amebic cultures resulted in low numbers of infected amebae (data not shown). Improved infection efficiency was seen when amebic trophozoites were allowed to attach to a filter membrane and the viral supernatant and polycation (polybrene) allowed to flow past the trophozoites. The efficiency of transformation was estimated following retroviral infection by counting hygromycin-resistant amebae at 4 days post-selection. Approximately 0.01% of the amebae could be selected by growth in 7.5 mg ml − 1 hygromycin. In contrast, 100% of uninfected amebae were killed by hygromycin concentrations \ 5 mg ml − 1 after 72 h growth at 37°C. The most conservative estimate of the efficiency of transformation would be 1× 10 − 7 (assuming a normal doubling time of 9 h [25]). DNA was extracted from an uncloned population of hygromycin-resistant cells and analyzed by PCR with primers specific for the proviral LTR. An amplicon of the predicted size (244 bp) was detected only in DNA extracted from pseudotyped vector-infected trophozoites, but not control samples (Fig. 1).
2.7. Southern hybridizations
3.2. Pro6iruses integrate into the genome of ameba trophozoites
For Southern blot analysis, E. histolytica genomic DNA was digested with the indicated restriction enzymes, electrophoresed through a 1% agarose gel, and transferred to GeneScreen (Dupont, Boston, MA) nylon membranes. Probes were radiolabeled using the Random Priming method (Prime-It II, Stratagene, La Jolla, CA). Hybridizations were carried out at 65°C in 0.5 M sodium phosphate, pH 7.0, 1 mM EDTA, 7% SDS and 1% bovine serum albumin (BSA). Filters were washed twice in 2× SSC/0.1% SDS at 50°C for 15 min, and then twice in 0.2 ×SSC/0.1% SDS at 50°C for 15 min. The washed membrane was exposed to radiographic film at −70°C overnight.
3. Results
3.1. Infection of trophozoites with pseudotyped retro6iral 6ector Incubation of amebae with virus in a static
To determine if the provirus was integrated into
Fig. 1. PCR detection of pantropic retroviral infection of E. histolytica. Primers amplified a 244-bp sequence of the proviral LTR from 1 mg of amebic DNA purified from two separate infections (lanes 1 and 2); negative control (lane N) and mock infected trophozoite DNA (lane M); and positive control DNA from 293/LSRNL producer cells (lane P). Lane L: 123-bp ladder for size marker.
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Fig. 2. Southern blot of DNA purified from E. histolytica following retroviral infection and hygromycin selection and probed with a HindIII/BglII fragment from pL(MCS)HHL containing the hyg gene. Panel A: KpnI -digested genomic DNA from two separate experiments with retroviral infections (lanes 1 and 2). KpnI-digested plasmid pLNAmActHL (lane P) and uninfected ameba DNA (lane N) are shown as controls. Panel B: HindIII -digested DNA with retroviral infection (lane 2) and with mock infection (lane 3), and a plasmid control (lane 1).
the amebic genome, DNA from infected trophozoites was subjected to restriction endonuclease digestion and Southern hybridization (Fig. 2). DNA extracted from retroviral-infected trophozoites was digested with KpnI, which has one recognition sequence in each LTR, or HindIII, which has a single recognition sequence between the 3%L21 and the LTR sequence. Digested DNA was electrophoresed on a 1.0% agarose gel, transferred to nylon, and hybridized to the HindIII – BglII fragment from pL(MCS)HHL containing the hyg gene [14]. A band corresponding the length of the entire provirus (4.2 kb) was detected in KpnI-digested genomic DNA derived from two separate infections (Fig. 2A). A smear of bands ( \ 3.7 kb) were found with HindIII-di-
gested DNA containing the provirus and amebic flanking sequences of variable lengths (Fig. 2B).
3.3. Pro6iral integration sites in E. histolytica DNA To determine the location of proviral insertion sites, hygromycin-resistant individual colonies were generated from single ameba cloned on soft agar containing 7.5 mg ml − 1 hygromycin B. Stable expression of the retrovirus-mediated hyg gene in the five clones was maintained for more than one year in the presence of hygromycin. After more than a month of culture in the presence of hygromycin, genomic DNA from five individual clonal populations was purified and analyzed by
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Southern hybridization and inverse PCR. The schematic drawing (Fig. 3) shows the resulting fragments amplified from the infected trophozoite DNA. The flanking regions (average of 200 nucleotides) from the five clones were sequenced, and the sequences contained the expected 2-bp deletion from the 5% end of the LTR (U3), which is characteristic of integration mediated by the retroviral integrase, followed by a novel sequence from the host E. histolytica DNA. Two clones were found to contain repetitive DNA from E. histolytica rRNA genes (Genbank accession X56991, X64142, X61116 and Y11272), confirming integration into the rRNA episome. Three clones did not show matches in database searches. Hybridization of uninfected E. histolytica DNA by slot blot with these three radiolabeled flanking sequences confirmed their origin from the amebic genome (data not shown). To determine the num-
ber of independent proviral integrations per ameba, a hyg gene fragment released from pL(MCS)HHL was gel-purified, radiolabeled, and hybridized to HindIII-digested genomic DNA from the five retroviral-infected, cloned ameba populations. Each clone had a single band of various size that hybridized to the probe, indicating integration of a single provirus at different sites in the amebic DNA (Fig. 4). These results confirm the first integration of foreign genes into the E. histolytica genome.
4. Discussion We have demonstrated integration and expression of foreign genes by retroviral-mediated gene transfer in E. histolytica. Pseudotyped pantropic retroviral particles containing an envelope with
Fig. 3. Inverse PCR amplification and sequence of retroviral integration flanking regions in E. histolytica clones. The deletion of two base pairs from end (U3) of the proviral DNA is characteristic of integrase-mediated insertion and is highlighted in bold. Sequences from five clonal populations are shown.
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Fig. 4. Integration of a single copy of proviral DNA demonstrated by Southern blot analysis of retrovirally infected E. histolytica clones. DNA samples from five clones (lane 1–5) were digested with HindIII and probed with the hyg gene fragment. Uninfected ameba DNA (lane N) is shown as a control. Molecular mass markers (in kb) are shown at the left.
the G glycoprotein of vesicular stomatitis virus (VSV-G) were developed that contain the E. histolytica actin promoter driving expression of the hygromycin phosphotransferase gene as a selectable marker. We found that simply allowing virus to flow past trophozoites on filters (approximately 10 min direct contact) produced the most reproducible infection. Growth of trophozoites in the presence of hygromycin (7.5 mg ml − 1) required expression of the introduced hygromycin phosphotransferase gene, as uninfected or mock-infected trophozoites were killed by concentrations as low as 5.0 mg ml − 1 [6]. The relatively low concentrations of hygromycin tolerated by the infected ameba, 7.5 versus up to 15 mg ml − 1 for ameba transiently transfected with hygromycin phosphotransferase sequences [6,7] most likely reflects the integration of only a single gene per ameba genome. Expression of hygromycin phosphotransferase demonstrates that the amebic actin promoter can mediate foreign gene expression in trophozoites. Definitive proof of integration was obtained by sequencing the regions flanking the integration sites. In three of five clones, the provirus integrated into unsequenced regions of amebic DNA, which did not appear to be in open reading frames. In two clones, the provirus integrated into repetitive DNA on the large extrachromosomal element
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(24.5 kb), encoding the small subunit (16S-like) ribosomal RNAs. The rRNA genes of E. histolytica are highly repeated on the large episome that comprises 10% of the parasite genome [26]. Therefore, integration occurs in both coding and noncoding regions of the genome, as described in insertional mutagenesis studies of zebrafish, where approximately 1/70 insertions results in an embryonic or larval mutant pheotype [28,29]. Previous studies showed that retroviruses preferentially integrate close to DNase 1-hypersensitive sites, thus chromatin structure favors acceptors sites for proviral integration in transcriptionally active regions of the DNA [30,31]. Examination of five proviral insertions into ameba DNA revealed no obvious integration site biases. Optimization of cloning methods for Entamoeba and evaluation of a larger number of recombinants will be required to address the important question of whether the entire genome could be targeted by this technique and whether a second retrovirus vector could integrate into an already infected cell, like in insect and mammalian cells with the pantropic vectors (unpublished data, J.C. Burns). The ultimate effectiveness of any transformation system depends on the ploidy of the organism. To date, the ploidy of E. histolytica has not been completely resolved. Several transfection systems have been designed that allow selection of hygromycin or G418 resistance in E. histolytica [2–7]. Two groups obtained transformation of E. histolytica trophozoites, in which the introduced sequences replicated episomally as unrearranged circular plasmids [2,3]. Both groups developed an inducible gene expression system using the tetracycline repressible operon, which should be useful for the expression of potentially toxic gene products [6,7]. The copy number of the plasmid could be increased with higher levels of hygromycin (up to 60 mg ml − 1), and 200-fold induction of reporter gene products were obtained [6,7]. In other experiments, expression of a lysine-rich 30 kDa surface antigen of Entamoeba dispar was inhibited by introducing antisense RNA sequences in a pEhActNeo shuttle vector [27]. Despite these important developments, no
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sequence had been successfully integrated into the amebic genome. The ability of retroviral vectors to integrate into the genome of infected cells has been exploited for gene therapy and for studies of insertional mutagenesis. Large-scale screens in zebrafish have identified many essential genes [28,29]. Genes mutated by proviral insertion can be readily cloned, because the integrated exogenous DNA provides a ‘tag’ for the mutated gene. Because retroviral vectors can accommodate between 10 and 13 kb of heterologous sequence, vectors can be constructed that contain coding regions for genes of interest in addition to marker genes (e.g. hyg) under the control of different promoters. The ability to stably transform protozoa has been a major advance in understanding these important pathogens. Such techniques will be particularly important in the study of amebiasis. Recent studies have confirmed that E. histolytica and E. dispar are genetically separate species [32,33]. Only E. histolytica is capable of invasion, but genes encoding potential virulence factors such as cysteine proteinases [33,34], galactose-inhibitable lectins [35], and amebapores [36] are also present in noninvasive E. dispar. Therefore, the ability to knock-out genes or overexpress others will be critical in understanding the genetic basis of virulence. We conclude that heterologous DNA can be expressed in E. histolytica trophozoites following infection with retroviral particles containing E. histolytica specific promoters. To our knowledge, this is the first report of genomic integration of foreign DNA in E. histolytica. Pantropic pseudotyped retroviral vectors should provide a powerful tool to deliver useful marker genes into trophozoites. The ability to introduce and express genes or perform insertional mutagenesis in amebae with retroviral vectors will permit genetic analysis and potentially lead to a better understanding of mechanisms of virulence. In addition the ability of pseudotyped mammalian retroviruses to integrate into a eukaryotic protozoan suggests that these vectors may be useful in other distantly related eukaryotes without established transformation systems.
Acknowledgements The authors wish to thank Barbara L. Sullivan for tissue culture and Felipe Olvera for plasmid manipulations. This work is supported in part by grants from the National Institutes of Health AI28035 (SR), DK35108 (SR), AI37671 (JCB), UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases nos. 950469 (JCB), and DGAPA-UNAM 207097 (AA).
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