ABSTRACT
Entamoeba histolytica, a protozoan parasite, is an important human pathogen and a leading parasitic cause of death. The organism has two life cycle stages, trophozoites, which are responsible for tissue invasion, and cysts, which are involved in pathogen transmission. Entamoeba invadens is the model system to study Entamoeba developmental biology, as high-grade regulated encystation and excystation are readily achievable. However, the lack of gene-silencing tools in E. invadens has limited the molecular studies that can be performed. Using the endogenous RNA interference (RNAi) pathway in Entamoeba, we developed an RNAi-based trigger gene-silencing approach in E. invadens. We demonstrate that a gene's coding region that has abundant antisense small RNAs (sRNAs) can trigger silencing of a gene that is fused to it. The trigger fusion leads to the generation of abundant antisense sRNAs that map to the target gene, with silencing occurring independently of trigger location at the 5′ or 3′ end of a gene. Gene silencing is stably maintained during development, including encystation and excystation. We have used this approach to successfully silence two E. invadens genes: a putative rhomboid protease gene and a SHAQKY family Myb gene. The Myb gene is upregulated during oxidative stress and development, and its downregulation led, as predicted, to decreased viability under oxidative stress and decreased cyst formation. Thus, the RNAi trigger silencing method can be used to successfully investigate the molecular functions of genes in E. invadens. Dissection of the molecular basis of Entamoeba stage conversion is now possible, representing an important technical advance for the system.
INTRODUCTION
Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death worldwide. The disease is prevalent in countries with poor sanitary conditions and leads to ∼50 million cases of invasive disease per year, resulting in 100,000 deaths annually (1, 2). The parasite has a two-stage life cycle: the ameboid trophozoites, which invade tissue, and infectious cysts, which are spread by contaminated food and water. Cysts that are ingested pass through the stomach and eventually excyst to trophozoites in the small intestine. The motile trophozoites are the invasive forms that cause disease symptoms, including colitis and liver abscess (3, 4). Due to an unknown stimulus, some trophozoites convert to cysts and are excreted in the stool. These cysts survive in the environment and transmit infection to subsequent hosts. Developmental switching is thus an essential feature of parasite biology, with interconversion from trophozoites to cysts responsible for both disease causation and disease transmission (5, 6). Despite efforts by many groups, in vitro encystation has not been possible in E. histolytica, which has made it difficult to study the encystation process (7).
Study of Entamoeba development has relied on the use of Entamoeba invadens, which causes clinical disease in reptiles similar to disease in humans (8). In E. invadens, both encystation (trophozoite-to-cyst induction) and excystation (cyst-to-trophozoite conversion) are highly efficient and synchronous under laboratory conditions (6, 9–11). Thus, E. invadens is a key organism to study the molecular mechanisms underlying the developmental switch in Entamoeba. Nonetheless, the use of E. invadens as a system has been limited by lack of genomic data and methods for genetic manipulation. However, significant recent advances, including genome reannotation, metabolic and transcriptome profiling of encystation and excystation, and development of transfection methods, have opened the door for detailed molecular studies in E. invadens (12–16).
In Entamoeba, methods for gene downregulation have been limited by the ploidy of the parasite and lack of homologous recombination. Despite these hurdles, a number of genetic approaches are available in E. histolytica, including tetracycline-regulated antisense (AS) silencing, use of dominant-negative approaches, destabilization domain protein regulation, and gene silencing via RNA interference (RNAi) (17–20). RNAi approaches include feeding the parasite bacteria expressing double-stranded RNA to a gene of interest, soaking parasites in small RNAs (sRNAs), use of short hairpin RNAs (sRNAs), sRNA generation by dual-promoter vectors, and gene silencing in the G3 strain (21–26). These techniques, although helpful, vary widely in their efficiency of downregulation and long-term silencing stability, as loss of silencing has been observed (27). Additionally, achieving silencing using shRNA is labor-intensive (22).
RNAi is a basic biological process regulating gene expression in multiple systems and has also become a standard biotechnology tool (28–32). The core pathway involves multiple proteins, but key to the RNAi pathway is the presence of small RNAs that associate with Argonaute proteins to mediate cognate gene silencing (33–35). E. histolytica has a robust and complex RNAi pathway that is mediated by 27-nucleotide (nt) sRNAs that associate with the E. histolytica Argonaute 2-2 protein to mediate transcriptional gene silencing (36–38). The sRNAs have 5′ polyphosphate termini and are similar to secondary sRNAs generated in nematodes (39, 40). The genome of E. invadens reveals the presence of several genes that code for the RNAi machinery, including four genes that encode full or partial Argonaute proteins, two genes that encode RNA-dependent RNA polymerase (RdRP), and a gene with an RNase III domain-containing protein (41). The presence of these genes indicates that the RNAi pathway may be functional in E. invadens. Furthermore, recent work has identified the presence of ∼27-nt sRNAs in E. invadens with features similar to those of E. histolytica sRNAs, including 5′ polyphosphate (polyP) termini and an association of 27-nt sRNAs with silenced genes (41).
We have recently developed a novel RNAi-based method to silence genes in E. histolytica that shows great promise. In this approach, a gene that has abundant endogenous antisense sRNAs can serve as a “trigger” to mediate silencing of another gene (20). Fusion of 132 bp of the coding region of a “trigger gene” to a full-length coding region of another gene results in generation of antisense sRNAs to the fused gene and subsequent gene silencing. This technique has been successfully used to downregulate the expression of several amebic genes, including virulence genes and transcription factors (20, 42). However, not all genes are amenable to silencing, as we noted that RNAi pathway genes, including genes that encode Argonaute (Ago2-1, Ago2-2, and Ago 2-3) and RNase III, were not silenced via the trigger method despite generation of functional sRNAs to the trigger-fused gene (43). The mechanism of trigger-mediated silencing is via transcriptional gene silencing and induction of histone modification, specifically dimethylation of lysine 27 of amebic H3 (38). Overall, the trigger-mediated RNAi silencing approach has a number of advantages, including robust silencing of the target gene and maintenance of silencing despite removal of the drug-selectable marker (20).
In this paper, we demonstrate advancement of the trigger silencing method with application to E. invadens. We demonstrate that genes with abundant antisense sRNAs in E. invadens can function as a trigger to mediate RNAi-based silencing. Silencing is mediated via the generation of antisense sRNAs to the trigger-fused gene. Importantly, trigger-mediated gene silencing is maintained even when the parasites undergo developmental switching between trophozoites and cysts. This trigger-based approach was used to silence two endogenous E. invadens genes and gave a predictable phenotype. This work is the first demonstration of gene silencing in E. invadens. Thus, the trigger silencing method can be used to study the complex molecular mechanisms involved in stage switching. This significant technical advance lays the foundation for future genetic studies in E. invadens and elucidation of the molecular networks that regulate Entamoeba development.
MATERIALS AND METHODS
Parasite growth, transfection, and induction of stage conversion.An E. invadens (IP-1) culture was grown at 25°C under standard conditions (15). To establish stable transgenic lines, the parasites were transfected with 50 μg plasmid by electroporation as previously described (12). The stable cell lines were maintained at G418 concentrations of 40 μg/ml or 80 μg/ml. Encystation was induced by incubation in 47% LYI-LG (supplemented with 7% adult bovine serum) as previously described (11). Encystation efficiency was calculated by the percent parasite survival after treatment with 0.1% Sarkosyl for 30 min at 4°C. For excystation, the encysted parasites were incubated overnight in distilled water at 4°C to lyse the trophozoites. The parasites were then excysted by the addition of bile salts and sodium bicarbonate in LYI-LG for 8 h (44).
Plasmid construction.The primers used in cloning are listed in Table 1. All the constructs used the plasmid pEi-CKII-Luc, in which the reporter gene is driven by the casein kinase II (CKII) promoter, as the backbone (12). Two genes were identified as potential trigger genes: EIN_003840 and EIN_152860. For EIN_003840 (referred to below as 003-Trigger), either 174 bp or 702 bp of the coding region (starting at the ATG start codon) was used as a trigger. For EIN_152860 (referred to below as 152-Trigger), 213 bp of the coding region starting at the ATG start codon was used as a trigger. All the trigger regions were cloned into pEi-CK-Luc via the NheI restriction site. For the 5′ trigger constructs, the full-length coding region of the gene to be silenced (luciferase; EiROM [EIN_255710] or EiMyb [EIN_241140]) was cloned downstream of the Trigger region at the NotI and AvrII sites. For the 3′ Trigger-Luc construct, the trigger was cloned via the SacII and SacI sites between the 3′ end of the luciferase gene and the CK 3′ regulatory region. All constructs were confirmed by sequencing. The Renilla luciferase gene was cloned into the pEi-Eno-Luc backbone at the XhoI-HindIII sites and served as a transfection control in transient-transfection assays.
Primers used in the study
Luciferase assays.Luciferase assays were performed in triplicate according to the manufacturer's protocol. For transient-transfection assays, cells were harvested 18 to 20 h after transfection and lysed in passive lysis buffer with addition of protease inhibitors (Promega E1300 lysis buffer, 1× HALT [Thermo Scientific]) (20). The cells were then subjected to sonication (one pulse) and assayed using the dual-luciferase kit for firefly and Renilla luciferase activity (Promega). Dual-luciferase assays were done as three independent experiments on separate days. For assaying luciferase from stable transfectants, cells were lysed in cell lysis buffer (Promega; E1300 lysis buffer) supplemented with protease inhibitor (1× HALT) and assayed using the firefly luciferase kit (Promega). Thirty micrograms of protein, as measured by the Bradford assay, was used in the luciferase assays and read using a luminometer to obtain relative light units (RLU). All stable-line luciferase assays were done as three independent experiments over a span of several days for both trophozoites and encysted parasites. Student's t test was used to determine the P value.
RNA extraction and RT-PCR.Total RNA was extracted from trophozoites using the mirVana kit (Ambion) according to the manufacturer's instructions. Total RNA was extracted from 24-hour cysts using TRIzol (Life Technologies). RNA was subjected to DNase treatment (DNase kit; Invitrogen) and reverse transcribed using oligo(dT) primers (Invitrogen). The resultant cDNA (3 μl) was used in subsequent PCRs (25-μl total volume). The number of PCR cycles was set to 30, and 10 μl of PCR products was run on a 1.5% agarose gel. The negative control (minus reverse transcriptase [RT]) was split away before the addition of Superscript RT (Invitrogen) and otherwise treated like the other samples. The primers used in RT-PCR are listed in Table 1.
Northern blot analysis.The small-RNA-enriched fraction was prepared using the mirVana kit (Ambion) following the manufacturer's protocol. High-resolution Northern blot analysis was performed as previously described (36) using 100 μg of sRNA-enriched fraction. Samples were run on a 15% polyacrylamide gel for sRNA analysis. All the primers used for Northern blot analysis are listed in Table 1. The primers were labeled using the mirVana miRNA probe construction kit (Ambion) following the manufacturer's protocol. The kit uses an in vitro transcription reaction to generate short radiolabeled RNA probes (labeled with α-ATP) that can be used to detect sRNAs.
Oxidative-stress and viability assay.The oxidative-stress and viability assay was performed as described previously (20, 45) with some modifications. The viability of E. invadens was assessed at various concentrations of H2O2 in order to determine an appropriate condition where the parasites were stressed (as determined by rounding and detachment) but still had ≥90% viability. H2O2 (4 mM) was established as optimal to induce oxidative stress. E. invadens trophozoites in log phase were subjected to 4 mM H2O2 for 1 h at 25°C. The tubes were viewed with a microscope to ensure rounding and detachment as an indicator of stress. The cells were then placed on ice for 10 min to detach the cells and spun down; the pellet was resuspended in 300 μl of LYI and mixed 1:1 with trypan blue immediately before cell counting under the microscope. Dead cells stain blue, while viable cells exclude the dye. The experiment was performed in duplicate on four different days. A t test was used to determine the P values. For RNA collection, cells stressed for 1 h at 25°C using 4 mM H2O2 were iced and spun down, and RNA was extracted using TRIzol (Ambion) following the manufacturer's instructions.
Encystation assayed in 96-well plates.The numbers of cysts of 152-Trigger-Luc (control) and the 152-Trigger-Myb were compared and analyzed using calcofluor white staining in a 96-well plate. Calcofluor white is a fluorescent stain that binds specifically to chitin present in the cyst wall and does not stain E. invadens trophozoites. Log-phase trophozoites were harvested, washed once with 50% LYI-LG, and resuspended in 50% LYI-LG (with 7% serum added). The cells were counted and seeded at a concentration of 50,000 cells per well. Trophozoites resuspended in LYI were seeded into wells and served as the negative control. The plates were sealed and incubated at 25°C for 48 h. At 48 h, the plates were spun down, and 20 μl of 50 μM calcoflour white (Sigma) in phosphate-buffered saline (PBS) was added to each well. The cells were imaged at ×10 magnification using ImageXpress Micro (Molecular Devices). The calcofluor white-stained cysts were viewed using a DAPI (4′,6-diamidino-2-phenylindole) filter, and a minimum of 44 images per well were taken using a laser and the image-based acquisition setting that automates the exposure time to take the best possible image. The images were then quantified using MetaXpress analysis software (Molecular Devices). The software automates counting of the cysts and provides the number of cysts stained with calcofluor for each well. The experiment was repeated on three different days; a t test was used to analyze the P values.
Statistical analysis.Student's t test was used to determine P values, and data with P values of <0.05 were determined to be statistically significantly different from each other for all the relevant experiments.
RESULTS
Identification of RNAi trigger genes in E. invadens.The emergence of E. invadens as a viable model organism in studying Entamoeba stage conversion necessitates having genetic tools to manipulate gene expression. We have recently shown in E. histolytica that genes with abundant sRNAs can serve as a trigger to silence a fused gene (20). Gene silencing occurs due to the generation of antisense sRNAs to the fused gene. Our recent work with sequencing the sRNA repertoire in E. invadens afforded us the opportunity to adapt the trigger method for use in E. invadens (41). In order to identify genes silenced endogenously by the RNAi pathway in E. invadens, we characterized the sRNA libraries from trophozoites and early-cyst (24-hour encystation), mature-cyst (72-hour encystation), and excysting (8 hours after induction of excystation) parasites. The sequences were mapped to the E. invadens genome using the Integrative Genomics Viewer tool (http://www.broadinstitute.org/igv). Similar to E. histolytica, there were abundant sRNAs (∼27 nt) that mapped to genes that had no or low mRNA transcript expression (37, 41). However, unlike E. histolytica, the E. invadens promoter regions had a greater number of sRNAs mapping to them, and there was a wider distribution of both sense (S) and antisense sRNAs over the length of a given gene's coding region. Keeping these considerations in mind, the genes selected as potential triggers fulfilled the following criteria: no mRNA expression in all life cycle stages but with a large number of sRNAs mapping to them. We selected two genes that met our criteria: EIN_003840 (1,284-bp coding region) had abundant antisense sRNAs in all life cycle stages (trophozoites, early cysts, mature cysts, and excysting parasites), and EIN_152860 (375-bp coding region) had both AS and S sRNAs in all life cycle stages (Fig. 1A and B). Both genes had low or no mRNA expression in all life cycle stages.
In silico identification of genes with abundant small RNAs and no mRNA expression and identification of potential trigger genes in E. invadens. (A) (Left) Schematic of the sRNAs that map to the EIN_003840 gene. The schematic displays ∼500 bp upstream and downstream of the coding sequence. The coding region of EIN_003840 is 1,284 bp, and RNA-Seq data show no mRNA expression. (Right) Graph showing the number of unique sRNAs that map to the gene at different time points of the parasite life cycle. Only antisense sRNAs map to EIN_003840. (B) (Left) Schematic of the sRNAs that map to the EIN_152860 gene. The schematic displays ∼500 bp upstream and downstream of the coding sequence. The coding region of EIN_152860 is 375 bp and shows no mRNA expression. (Right) EIN_152860 has both sense and antisense sRNAs mapping to it during all stages of the life cycle. ORF, open reading frame.
Trigger gene fusion silences the luciferase reporter gene.To identify whether the coding regions of EIN_003840 and EIN_152860 could function as triggers to mediate gene silencing, we fused 174 bp from the 5′ end of the coding region of EIN_003840 and 213 bp from the 5′ end of EIN_152860 to a luciferase reporter construct. These regions of the genes were picked because a number of antisense sRNAs target the regions. The fragments were fused to the 5′ end of firefly luciferase and driven by the CKII promoter (Fig. 2A). Firefly luciferase with no trigger fusion and driven by the CKII promoter served as the positive control, and a firefly luciferase construct without a promoter served as the negative control (12). Stable parasite transfectants were generated in which the plasmids are maintained episomally under G418 (40 μg/μl or 80 μg/μl) selection. We determined that in both trigger-luciferase fusions, there was significantly lower firefly luciferase activity than in control cell lines with no trigger (Fig. 2B). Similar results were obtained when the stable cell lines were subjected to 80 μg/ml of G418 (data not shown). Overall, both trigger genes resulted in low luciferase signal with statistically significant reduction compared to controls. Thus, similar to results obtained with E. histolytica, fusion of part of the gene to which abundant sRNAs map leads to reduction of firefly luciferase activity.
Fusion of the trigger region to luciferase results in reduced luciferase activity. (A) Schematic of the constructs used in the assay. A 174-bp region of EIN_003840 (003-Trigger-Luc) and 213 bp of EIN_152860 (152-Trigger-Luc) were fused upstream of the luciferase gene. ATG is indicated by the arrows, and the stop codon is indicated by the asterisks. All the constructs were driven by the casein kinase (CKII) promoter. Stable parasite lines with the above-mentioned constructs were generated, and luciferase activity was assayed. (B) The fusion of 174 bp of EIN_003840 and 213 bp of EIN_152860 upstream of the luciferase coding region significantly reduces (*, P < 0.005) luciferase activity compared to the positive control (CKII-Luc). The luciferase activity was normalized to protein as measured by Bradford assay. Experiments were performed on three separate days; averages are shown, and the error bars indicate standard errors of the mean (SEM). The P values were determined using a t test. (C) High-resolution Northern blot of the trigger fusion and the wild-type cell lines. An sRNA-enriched fraction (100 μg) was loaded and probed for AS sRNAs generated to the luciferase gene. AS sRNAs were detected in all of the trigger fusion stable lines, as indicated by the arrows. The loading control was endogenous sRNAs to EIN_104920. (D) The sRNAs generated to luciferase are functional, as demonstrated by transient-transfection assays of the stable lines with a luciferase reporter construct. The inset shows a schematic of the assay setup. The firefly construct is expressed well in the wild-type cells but is not expressed well in the stable line with a Trigger-Luc fusion (*, P < 0.05). There was robust Renilla luciferase activity in all the stable lines, with values always above 5 × 104 RLU, indicating that the AS sRNAs silence the firefly luciferase gene specifically. The values were normalized to Renilla luciferase activity; experiments were performed for at least three independent transient transfections, and the error bars indicate SEM. The P values were determined using a t test.
Antisense sRNAs generated by trigger-mediated silencing are functional to silence a reporter gene in trans.In E. histolytica, gene silencing using the trigger approach led to the generation of abundant ∼27-nt AS sRNAs to the fused gene (20). To determine if a similar phenomenon occurred in E. invadens, we performed high-resolution Northern blot analysis using strand-specific oligonucleotide probes to determine if we could detect sRNAs to the luciferase gene. A total of 100 μg of sRNA-enriched fraction from the wild type, 003-Trigger-Luc, and 152-Trigger-Luc were probed for AS sRNAs to luciferase. The presence of sRNAs that are ∼27 nt in length in the trigger-luciferase fusion stable cell lines mimics the endogenous small populations in E. invadens (Fig. 2C). This confirms that trigger-luciferase fusion in E. invadens generates sRNAs targeting luciferase, similar to what we have previously observed in E. histolytica (20).
To further confirm that the sRNAs generated to the luciferase gene are functional and can specifically silence firefly luciferase, we transiently transfected the Trigger-Luc stable cell lines with two constructs: one that expresses firefly luciferase and one that expresses Renilla luciferase. Since the sRNA silencing is sequence specific, there should be no impact on Renilla luciferase expression, but there should be silencing in trans of the firefly luciferase (20). We identified that in the stably transfected cell lines 003-Trigger-Luc and 152-Trigger-Luc, a newly transfected firefly luciferase construct was silenced compared to robust expression of a Renilla luciferase construct (Fig. 2D). In wild-type cell lines, both constructs were expressed. Thus, the sRNAs generated by the trigger-luciferase fusion mediated sequence-specific silencing, as indicated by the reduction in firefly luciferase but lack of impact on Renilla luciferase expression. The Renilla luciferase activity was above 5 × 104 RLU for the stable lines during the three trials, indicating that trigger silencing is specific to firefly luciferase. This demonstrates that the sRNAs generated in the trigger-luciferase fusion are functional, as evidenced by their ability to silence a homologous copy of firefly luciferase in trans.
In E. histolytica, loss of the trigger plasmid occurs relatively rapidly after removal of drug pressure (within 1 to 2 weeks). Once the trigger plasmid is lost, the luciferase sRNAs are also lost, and a transient transfection with the luciferase construct results in wild-type levels of luciferase activity (20). We determined that in E. invadens the plasmid is lost very slowly and that the trophozoites harbor some plasmid even after a year without the drug (data not shown). Thus, a parallel experiment with loss of plasmid, loss of sRNA, and loss of silencing is not possible in E. invadens.
Gene silencing occurs independently of the position of the trigger region.In E. histolytica, the effect on gene silencing was independent of the location of the trigger sequence, and the trigger could be placed at either the 5′ or the 3′ end of a reporter gene (20). To examine the effect of the location of the trigger in E. invadens, we fused 702 bp from the coding region of EIN_003840 (702bp-003-Trigger-Luc) as the trigger region to the 5′ or 3′ end of the luciferase gene (Fig. 3A). Stable transfectant lines (maintained with 40 μg/μl of G418) were generated, and firefly luciferase activity was measured. Irrespective of the position of the trigger region (5′ trigger or 3′ trigger), luciferase activity was reduced (Fig. 3B). Northern blot analysis for luciferase AS sRNAs indicated the generation of AS sRNAs in both the 702bp-003-Trigger-Luc and 702bp-003-Luc-Trigger stable lines (data not shown). The presence of abundant AS sRNAs generated to the reporter gene signifies the functionality of the trigger at both the 5′ and 3′ ends of a reporter gene. This suggests that, similar to E. histolytica, the trigger region can function to mediate gene silencing at either at the 5′ end or the 3′ end of the gene, indicating that this method of gene silencing is independent of the position of the trigger region.
Silencing of the reporter gene construct is independent of the location of the trigger. (A) Schematic of the constructs used in the assay. ATG is indicated by the arrows, and the stop codon is indicated by the asterisks. (B) Luciferase activity of the Trigger-Luc fusion is reduced irrespective of the position of the trigger. The experiment was performed in triplicate (*, P < 0.05, as determined by a t test; the error bars indicate SEM).
Trigger-mediated gene silencing is maintained during encystation and excystation.As the parasite undergoes a developmental shift from vegetative growth to a quiescent stage (trophozoites to cysts), approximately half of the E. invadens genome is differentially expressed at some point during the stage transition (15). Thus, it is imperative to determine if silencing is stable during this massive change in cell physiology. Given that the E. invadens sRNA profile remains unchanged during the interconversion between trophozoites and cysts, we presume that the silencing will persist (41). To determine if trigger-mediated gene silencing is stable under both encystation and excystation conditions, the cell lines 003-Trigger-Luc and 152-Trigger-Luc were encysted, and the firefly luciferase activity was measured at two different time points: 24 hours (early cysts) and 72 hours (mature cysts). The encystation efficiency was estimated by calculating the percent survival of the mature cysts (72 hours of encystation) after treatment with 0.1% Sarkosyl for 30 min at 4°C. The overall encystation efficiency was calculated to be ∼40 to 60% for the different stable lines. The wild-type parasites and the stable line harboring the CKII-Luc plasmid (at a drug concentration of 40 μg) served as the control strains.
In trigger-luciferase cell lines, the silencing was stably maintained in both early cysts and mature cysts, as shown by the significant reduction in luciferase activity compared to the CKII-Luc cultures (Fig. 4). However, we observed a difference in the extent or stability of silencing between 003-Trigger-Luc and 152-Trigger-Luc. When encysted, the 003-Trigger-Luc parasites showed a slight increase in luciferase activity compared to the activity observed in trophozoites (Fig. 4, inset). This increase in the luciferase activity of 003-Trigger-Luc is statistically significant. The leakiness could be attributed to the amount of AS sRNAs specifically targeting 003-Trigger-Luc and 152-Trigger-Luc. A larger amount of AS sRNAs map to the 213-bp trigger region in 152-Trigger-Luc than to the 174-bp trigger region in 003-Trigger-Luc. However, overall, the luciferase activities of both constructs are significantly reduced compared to that of the positive-control CKII-Luc. Thus, silencing is stably maintained by the trigger method even when the cells are developmentally triggered to encyst, with 152-Trigger being a more stable silencing vector.
Silencing mediated by Trigger-Luc fusion is stable during the developmental switch from trophozoites (Trophs) to cysts and excystation from cysts to trophozoites. Stable Trigger-Luc lines were induced to encyst and excyst, and luciferase activity was monitored at 24 hours (early cyst), 72 hours (mature cyst), and 8 hours of excystation. The luciferase activity was reduced in the Trigger-Luc line at the various time points compared to the positive control (CKII-Luc). The average encystation efficiencies for wild-type, CKII-Luc, 003-Trigger-Luc, and 152-Trigger-Luc stable lines (as measured by Sarkosyl resistance at 72 h) were 42.11%, 44.67%, 56.75%, and 50.37%, respectively. Experiments were performed on three different days; the error bars indicate SEM. *, P < 0.05. The P values were determined using a t test.
To further evaluate the stability of trigger silencing in excysting parasites, the stable lines were induced in excystation medium and luciferase activity was measured 8 hours after induction of excystation. The trigger-luciferase stable lines maintained luciferase silencing even when excysted (Fig. 4). The developmental switching from trophozoites to the cyst phase requires a multitude of factors and genes to be turned on/off. The stability of trigger-induced silencing during this developmental cascade renders this a viable tool for gene silencing in all life cycle stages.
Silencing an endogenous E. invadens gene is achieved using the trigger approach.The successful downregulation of the luciferase gene and sRNA detection led us to investigate whether we could achieve gene knockdown of a chromosomally encoded E. invadens gene. We assayed two genes: a rhomboid protease gene and a Myb transcription factor gene. 152-Trigger was chosen to silence these genes due to the stability of its trigger silencing under encysted conditions.
The first gene tested, EIN_255710 (EiROM), is a single-copy gene that is a homolog of an E. histolytica rhomboid protease 1 gene (EHI_197430 [ROM1]). Rhomboid proteases are known virulence factors that function in adhesion and phagocytosis of the host cells and thus have a well-studied role in host-parasite interactions (46–48). By fusing 152-Trigger to full-length myc-tagged ROM1 (1,008-bp coding region), the 152-Trigger-EiROM construct was generated (Fig. 5A); stably transfected parasites were generated and maintained under drug pressure (G418, 40 to 80 μg/ml). To determine if there is reduction in the transcript level, semiquantitative RT-PCR was performed on RNA extracted from trophozoites and early (24-hour) cysts from stable lines maintained at 80 μg/ml G418. The abundance of the EiROM transcript was reduced compared to the wild-type control (Fig. 5B). EIN_192230 was the loading control and showed equivalent cDNA loading in the PCRs. Thus, the presence of the trigger fused to EiROM leads to a reduction in both the episomal and chromosomal transcript levels, as evidenced by the results of the RT-PCR. To confirm that the transcript reduction is maintained when parasites are encysted, RT-PCR was performed on RNA extracted from 24-hour cysts; the data indicate that the transcript reduction of the gene is continuously maintained in encysted parasites (Fig. 5B). This reiterates the usefulness of the trigger approach during the different stages of the parasite life cycle. The overall reduction of EiROM1 with the trigger method, even though substantial, was not complete, which is very different than the level of silencing we noted in E. histolytica. This could be due to various factors and differences between the two species, including promoter strength, more active degradation of small RNAs, and generation of some amount of small RNAs that cannot actively silence the gene in E. invadens.
Gene knockdown is achieved by fusing 152-Trigger to the full-length EiROM gene. (A) Schematic of the 152-Trigger-EiROM construct used to generate stable lines. The full-length (1,008-bp) region of EiROM was utilized. (B) RT-PCR to detect ROM transcript in the wild-type and 152-Trigger-ROM (G418, 80 μg/ml) stable lines indicates a reduction in transcript level in the Trigger-ROM stable line, with reductions observed in both trophozoites and 24-hour cysts. EIN_192230 is the loading control; EIN_099680 is the encystation control and is upregulated when parasites are encysted. Specificity of silencing to the ROM gene is demonstrated by an RT-PCR that shows unchanged transcript levels of genes with 42% (EIN_057990) and 37% (EIN_219350) sequence similarity to EiROM in the Trigger-ROM stable parasites compared to wild-type controls. (C) High-resolution Northern blot analysis probing for AS sRNAs generated to ROM showed abundant sRNAs compared to the wild-type control at drug (G418) concentrations of 40 μg/ml and 80 μg/ml.
To determine if silencing is sequence specific, we performed RT-PCR to detect transcripts for EIN_057990 (EiROM3) and EIN_219350 (EiROM4) that have 42% and 37% sequence similarity to EiROM1. The mRNA level was unaffected for both of the genes, confirming that gene silencing via the trigger approach is sequence specific in both trophozoites and cysts (Fig. 5B). Northern blot analysis detected the presence of AS sRNAs to EiROM, further confirming that trigger silencing was mediated by sRNAs (Fig. 5C).
In order to determine the broader generalizability of the trigger approach, we utilized the trigger to silence a second gene encoding a Myb homolog (EIN_241140) (Fig. 6A). This Myb protein is a member of the SHAQKY family of Myb transcription factors and controls the expression of a subset of amebic genes involved in the process of encystation (49, 50). We fused the full-length Myb gene to 152-Trigger and generated stable transfectants (G418, 40 to 80 μg/ml). In trophozoites, no expression is evident for either the wild-type or 152-Trigger-Myb cell line (G418, 80 μg/ml); however, in 24-hour cysts, when the Myb gene is upregulated, one can detect transcript reduction from the 152-Trigger-Myb cell line (Fig. 6B). To further confirm that the trigger approach did not modulate the transcript levels of other proteins encoded by the Myb family of genes, we performed RT-PCR to detect a transcript for EIN_277760. The EIN_277760 transcript was unaffected in the downregulated (EIN_241140) stable line, suggesting that the trigger approach is specific to the gene that is fused to the trigger.
Gene knockdown is achieved by fusing EiMyb to 152-Trigger. (A) Schematic of the 152-Trigger-EiMyb construct used in establishing stable cell lines. The full-length (546-bp) portion of the Myb gene was cloned downstream of 152-Trigger, resulting in a 152-Trigger-EiMyb construct. (B) RT-PCR demonstrating transcript reduction of the Myb homolog (EIN_241140) when parasites were induced to encyst (G418, 80 μg/ml). The loading control indicated equivalent loading. The encystation control (EIN_099680) was upregulated when encysted and served as a control to indicate optimal encystation. (C) RT-PCR analysis of 152-Trigger-Luc (G418, 80 μg/ml) in unstressed versus stressed parasites (4 mM H2O2; 1 h at 25°C) showed upregulation in the EiMyb transcript level when exposed to H2O2 stress. The loading control (EIN_192230) showed equivalent loading of cDNA. The unchanged transcript level of EIN_277760, a gene that encodes a Myb family protein, demonstrated specificity of silencing. (D) Viability of H2O2-stressed parasites compared to 152-Trigger-Luc (control parasites) as assessed by trypan blue staining. Under no-stress conditions, there was no difference in the viabilities of the 152-Trigger-Myb parasites and the control line. Under 4 mM H2O2, there was decreased viability in the 152-Trigger-EiMyb stable lines compared to control cell lines. The results are averages of four independent biological experiments performed on 4 days. The errors bars indicate SEM; *, P < 0.05 (P values were determined using the t test). (E) Representative images of calcofluor-stained cysts and trophozoites of the 152-Trigger-Luc (control) and 152-Trigger-Myb parasite lines (G418, 80 μg/ml). The cells were imaged at ×10 magnification using the ImageXpress Micro (Molecular Devices). The cyst stains specifically with the fluorescent stain, while the trophozoites remain unstained. (F) The number of cysts of 152-Trigger-Luc (control) parasites was compared to that of 152-Trigger-Myb as determined by calcofluor staining and analysis using ImageXpress (equipped with a laser and image-based acquisition) in a 96-well format. The parasites were seeded at a concentration of 50,000 cells/well, induced to encyst, and analyzed using 50 μM calcofluor at 48 h. A minimum of 20 wells per parasite line per experiment were analyzed, and biological-replicate experiments were performed on three independent days. The error bars indicate SEM; the P value was determined using a t test (*, P < 0.05). 152-Trigger-Myb showed reduced cyst numbers compared to the control cell line.
In order to determine whether trigger-mediated transcript reduction was substantial enough to give an expected phenotype, we tested 152-Trigger-Myb (G418, 80 μg/ml) for survival under oxidative-stress conditions. Myb transcription factors are known to be upregulated as a response to oxidative stress, and we found that the gene is also upregulated during oxidative stress (Fig. 6C) (45, 49). We have previously demonstrated that downregulation of a Myb gene in E. histolytica caused a decrease in parasite viability under oxidative stress (20). Thus, we analyzed the EiMyb knockdown for a survival phenotype in response to H2O2. The control and 152-Trigger-Myb cell lines (G418, 80 μg/ml) were subjected to 4 mM H2O2 stress. The viability of the parasites was assessed using a microscope, and parasites were counted using trypan blue. The data demonstrate that reduction in EiMyb results in reduced parasite viability in response to H2O2 stress (Fig. 6D). Thus, despite the modest transcript reduction, a phenotype for survival under oxidative stress was observed.
Myb transcription factors are known to regulate encystation in Giardia lamblia and are required in different stages of development in Dictyostelium (51–54). The upregulation of EiMyb during encystation suggests a putative role for the transcription factor during the process of amebic encystation (49). To investigate if there is a phenotype when the EiMyb gene is downregulated (in the 152-Trigger-Myb cell line), we used a high-throughput encystation assay in 96-well plates with calcofluor staining as a readout of cyst formation. A 96-well plate was seeded with 50,000 trophozoites/well in low-glucose encystation medium for both the 152-Trigger-Luc (control) and the 152-Trigger-Myb strains (G418, 80 μg/ml). The plates were sealed and incubated for 48 h at 25°C. After 48 h, the cells were stained with calcofluor white, a fluorescent stain that binds to chitin, the main component of the cyst wall (55, 56). Calcofluor white specifically stains the cyst cell wall, while trophozoites do not stain (Fig. 6E). Images were taken using an automated microscope and quantitated. There was a significant decrease in cyst numbers in the 152-Trigger-Myb stable cell line compared to the control. The decrease in cyst numbers in the 152-Trigger-Myb cell line confirms an important role for EiMyb in encystation (Fig. 6F). Thus, despite the modest transcript reduction, a phenotype of reduced encystation was observed when the EiMyb gene was downregulated. Further studies to more carefully delineate the roles of EiMyb in amebic biology will need to be done; however, our work demonstrates proof of concept that gene downregulation is possible, with the concomitant expected phenotypes.
The successful reduction in the transcript levels of the two E. invadens genes demonstrates the validity of the tool in silencing multiple amebic genes in E. invadens. The data here indicate (i) that endogenous genes can be downregulated in E. invadens using the trigger gene-silencing approach, (ii) that this downregulation is stable during the transition between trophozoites and cysts, (iii) that the silencing is sequence specific, and (iv) the feasibility of using the trigger-based silencing approach to study the functions of amebic genes.
DISCUSSION
E. invadens is the model system to study developmental control in Entamoeba species. However, the lack of tools to study genetic processes has significantly hampered research efforts in the organism. Entamoeba has a complex endogenous RNAi pathway mediated by 27-nt sRNAs that mediate transcriptional gene silencing (36). Here, we describe our successful efforts at harnessing the endogenous RNAi machinery to develop a tool for genetic manipulation in E. invadens. This trigger-based gene-silencing method has several advantages, including long-term stable silencing; maintenance of silencing during developmental-stage conversion, including both encystation and excystation; and sequence specificity. We demonstrated successful downregulation of two genes, EiROM1 and EiMyb, and successfully demonstrated that EiMyb knockdown has a phenotype of reduced survival under oxidative stress and reduced encystation efficiency. Overall, this work represents a significant advance for the field, and the trigger silencing tool will be valuable to study the molecular mechanisms of gene regulation involved in the developmental cascade.
The ability to induce encystation and excystation under standard laboratory conditions makes E. invadens an attractive model to study the molecular mechanisms associated with the developmental processes. Thus, the stability of silencing during the multitude of developmental changes is paramount in the use of this tool in different life cycle stages of the parasite. Recent transcriptome-sequencing (RNA-Seq) data from E. invadens has uncovered the fact that the sRNA repertoire does not change during the different developmental stages of the parasite (15). Therefore, we unfortunately did not find genes that could function as stage-specific triggers. However, we know that trigger silencing is dependent on promoter activity (20), and thus, the recent identification of stage-specific promoters in E. invadens means that we can use this approach to develop stage-specific trigger silencing (57). Expressing the trigger under the stage-regulated promoters will help regulate the exact stage at which transcription occurs and at which small RNAs can be generated. For example, a trophozoite-specific promoter can be used to generate sRNAs targeted to a particular gene of interest only in the trophozoite stage. This would be useful in studying the developmental cascade of Entamoeba and adds to the functionality of the trigger approach.
We observe that even though both triggers maintain silencing when encysted, 003-Trigger-Luc exhibits some leakiness compared to 152-Trigger-Luc. The silencing in the 003-Trigger-Luc parasite line is restored when parasites are excysted, suggesting that some factor during the process of encystation leads to its instability. The leakiness could be attributed to the fact that (i) the trigger-to-gene length ratio is lower in 003-Trigger-Luc (a 174-bp trigger region compared to the full coding region of 1,284 bp), and hence, fewer antisense sRNAs map to 003-Trigger-Luc, or (ii) the presence of a mixed population of sense and antisense sRNAs makes 152-Trigger-Luc a more effective silencing trigger. Sense sRNAs have been shown to have a role in posttranscriptional gene silencing in plants in a Dicer-dependent manner (58). However, the role played by sense sRNAs in Dicer-independent gene silencing is unknown in Entamoeba and warrants further investigation. The exact mechanism of the amplification pathway and generation of sense and antisense sRNAs in Entamoeba is unknown. In Caenorhabditis elegans and plants, sRNAs are amplified via an RNA-dependent RNA polymerase that uses the mRNA as the template (40, 59–61). These sRNAs have the ability to extend to distant cells in C. elegans and hence modulate functions in the distant cells (62, 63). Endogenous sRNAs in Entamoeba harbor properties similar to those of secondary sRNAs of C. elegans, including 5′ polyphosphate termini and AS nature (36). The elucidation of the key players in the RNAi-mediated gene-silencing machinery will lead to development of more tools that harness the power of RNAi.
The mechanism of sRNA-mediated gene silencing is slowly being unraveled in Entamoeba and suggests a link between sRNAs and histone modification. In the E. histolytica G3 strain, gene silencing is associated with a significant enrichment of histone H3 at the silenced loci, and AS sRNAs are directly linked to mediating transcriptional gene silencing (64). We have recently shown that one of the ways that the AS sRNAs mediate silencing is via a repressive histone methylation mark in lysine 27 of histone H3 (38). However, in E. histolytica, genes in the RNAi pathway, including the Argonaute 2-2 gene, could not be silenced via the trigger method, even though sRNAs were successfully generated by trigger gene fusion (43). Whether a similar mechanism of gene silencing or identification of genes that cannot be silenced will also be noted in E. invadens remains to be determined.
There is a difference between the quality of silencing achieved by the trigger-based approach in E. histolytica and that in E. invadens. Trigger gene silencing in E. histolytica is more robust than in E. invadens. One important difference is the greater abundance of sRNA mapping to the promoter region of a silenced gene in E. invadens (41). Small RNAs targeting the promoter region have been shown to lead to silencing in plants, fission yeast (Schizosaccharomyces pombe), flies, and mammalian cells (65–67). Thus, it will be interesting to investigate if a portion of the promoter region to which a large number of AS sRNAs map would enhance the trigger silencing we have observed to date in E. invadens. Also, in E. histolytica, there is a significant bias of the sRNAs to the 5′ end of the gene (20), while the sRNAs in E. invadens span the coding sequence and no 5′ bias is observed (41). This 5′ bias of the sRNAs could make the RNAi pathway, which is potentially RdRP dependent, more robust in E. histolytica. Another difference is the presence of a slightly larger species of sRNAs (∼31 nt), together with the ∼27-nt species of sRNA observed in E. invadens. The significance of the larger species of RNA and their role in gene silencing warrants further investigation. Additionally, there could be a difference in the dynamics of epigenetic memory mediated by the RNAi pathway in E. histolytica versus E. invadens that makes the RNAi machinery much more robust.
In conclusion, we describe an adaptation of RNAi-based trigger gene silencing to E. invadens and demonstrate its ability to downregulate genes and to give a predictable phenotype. This offers a reliable method of gene knockdown and opens up avenues of further investigation of Entamoeba developmental biology.
ACKNOWLEDGMENTS
We thank all members of the Singh laboratory for productive discussions.
Funding was provided by NIH grants R21-AI102277 and R21-AI117171 to U.S.
FOOTNOTES
- Received 14 September 2015.
- Returned for modification 31 October 2015.
- Accepted 14 January 2016.
- Accepted manuscript posted online 19 January 2016.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
