We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.
Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.
Cystic echinococcosis (CE) is one of the most important helminth diseases caused by Echinococcus granulosus, a small tapeworm within the family Taeniidae1,2. Extensive studies on immunodiagnostic and vaccine development for E. granulosus have been carried out. However, inadequate knowledge about the molecular basis of parasite biology poses major limitations in the diagnosis, management, and prevention of hydatid disease3,4,5,6.
In recent years, due to the development of genome sequencing and transcriptomic methods, a wide range of molecular studies have been conducted on flatworms by several research groups7,8,9. However, in the world of parasites, advances in gene transfer technology in parasitic flatworms are still limited compared with the highly reproducible transient transduction methods developed for some protozoa10,11,12.
The use of viral delivery systems has emerged as an essential tool for transgene delivery and gene/protein investigations over the last two decades13. Lentivirus infects both dividing and non-dividing cells, thus making it possible to infect postmitotic cells14,15,16. Recent evidence indicates that using a lentivirus-based transduction system in mammalian cells offers the potential to overcome most of the limitations of previous knock-in/knock-down techniques. The design and construction of expression lentiviral vectors with appropriate molecular markers, such as GFP expression, have been described previously16. Therefore, we evaluate lentiviral transient transduction of a GFP reporter gene in the protoscoleces and strobilated worms of E. granulosus.
This study was approved by the National Institute for Medical Research Development and the Research Ethics Review Committee, No. 958680. Lentiviruses are classified as BSL-2 organisms; hence, all laboratory culture procedures in this protocol were carried out using sterile laboratory practices and conducted under a laminar flow hood according to NIH guidelines. Figure 1 demonstrates a schematic presentation of the study protocol for the different E. granulosus stages.
1. Collecting hydatid cysts
2. Biphasic cultivation of PSCs of E. granulosus to obtain adult worms
NOTE: Isolated PSCs should be cultured in sterile conditions under a laminar flow cabinet Class II. Prepare the solid and liquid phases of the biphasic culture medium separately before proceeding to the next steps19.
3. Monophasic cultivation of PSCs of E. granulosus
4. Cell culture for virus production and preparation
NOTE: Human embryonic kidney 293T (HEK293T) cells were obtained for vector production from the Pathology and Stem Cell Research Center, Kerman University of Medical Sciences.
5. Production and preparation of the virus with the third-generation lentiviral vectors
NOTE: The lentivirus vector pCDH513b (transfer vector) and PLPII, PLPI, and PMD2G (helper vectors) were used to express the GFP reporter gene. See the Table of Materials and Supplemental Figure S1.
6. Transient transduction of different stages of E. granulosus with the virus
Here, we describe a rapid and efficient transient transduction technique in E. granulosus by using third-generation lentiviral vectors. We cultured PSCs in a biphasic culture medium to obtain strobilated worms, as described previously25,26. Protoscoleces develop into strobilated worms after 6 weeks in vitro. Different stages of E. granulosus were observed in the biphasic culture medium, including invaginated PSCs (Figure 2A), evaginated PSCs (Figure 2B), and strobilated worms with first and third proglottid formation (Figure 2C–E). Protoscoleces and the strobilated worms obtained from monophasic and biphasic cultures, respectively, were transfected with GFP-expressing lentiviruses (Figure 3). To avoid autofluorescence effects, microscopic observations were compared with the background fluorescence in all three groups of samples, and all samples above this background level were considered transfected.
We adjusted the field illumination and lowered the shutter to prevent any possible autofluorescence emission in the control samples for each treatment. We then checked lentiviral vector-treated samples, wherein green light emission against a black field was considered effective transient transduction. HEK293T cells-the transient transduction process control-clearly expressed GFP (Figure 3D). The adult worms expressed GFP most distinctly in the tegumental layer (Figure 3F); however, PSCs demonstrated a somewhat lower level of GFP expression after 48 h. Some cyst fluid residues and/or germinal layer debris around the PSCs caused some fluorescence in the background in the transfected samples. The intensity of fluorescence in HEK293T cells and strobilated worms increased after 24 h and 48 h (minor changes were observed in PSCs).
Figure 1: A schematic presentation of the study protocol. Abbreviation: PSCs = protoscoleces. Please click here to view a larger version of this figure.
Figure 2: The different stages of Echinococcus granulosus in biphasic culture medium. (A) Invaginated PSCs, (B) evaginated PSCs, (C, D) worms in the process of strobilation, (E) strobilated worms with third proglottid formation, (F) worms in different stages of strobilation in the culture medium. Scale bars = 200 µm. Please click here to view a larger version of this figure.
Figure 3: Transient transductionof different stages of Echinococcus granulosus and the control cell line in light and fluorescence microscopy. (A,D) HEK293T cells as transduction process control, (B, E) protoscoleces, and (C, F) strobilated worms. (G, H, I) Mixed light and florescence microscopy. Scale bars = 50 µm, 100 µm, and 500 µm. Please click here to view a larger version of this figure.
Supplemental Figure S1: Map of pCDH-CMV-MCS-EF1-GreenPuro cDNA cloning and expression vector. Please click here to download this File.
Understanding the molecular basis of nematodes and Platyhelminthes biology is crucial to understanding the pathogenicity of zoonotic parasites27. The lack of an effective gene evaluation system is a major obstacle to the direct interpretation of functional genetics of cestode parasites, including Echinococcus species12,27. The present study demonstrates the excellent potential of lentivirus in E. granulosus transient transduction.
Lentiviral transduction combines the simplicity of use and speed of transient transduction with the strong expression of stable cell lines to deliver transgenes to mammalian cells28. The major goal of this work was to illustrate the process of lentiviral vector transduction, which will be critical in future Echinococcosis research.
Specific critical points must be noted in this protocol. Strict sterile workflow conditions should be observed due to the omission of antibiotics in the culture medium, without which bacterial and fungal contamination can be expected after 24 h. Microbial contamination reduces the motility and viability of the parasites, and both transfected and control samples will be lost after 72 h.
Selecting the appropriate stages in the life cycle of Platyhelminthes and suitable culture conditions are key to successful gene transduction12,27. We tested our lentiviral transduction method in the metacestode and strobilated stages. The results showed that strobilated worms were more responsive to transient transduction of the gene than the PSCs, which may be the result of the extensive tegumental structure in the strobilated forms. However, due to the nature of lentiviral transduction, the fluorescence intensity in the multicellular PSCs and strobilated worms is typically much lower than in monolayer HEK293T cells26.
The potential of using Echinococcus as a new model in flatworms for studying biological phenomena has long been discussed. Therefore, a better understanding of Echinococcus biology as a model organism can be useful for expanding stem cell research and regulating the gene expression and evolutionary biology of other platyhelminths8,29,30,31,32. There is an urgent need for applied genetic research at the genomic and transcriptomic levels for Echinococcus and various parasitic and free-living flatworm model systems. Therefore, developing an effective gene transfer process for tapeworms paves the way for new techniques, such as virus-based gene knock-in or CRISPR-Cas-mediated gene editing32,33. This work presents lentiviral transduction in a tapeworm model and demonstrates promising outcomes, with potential implications for flatworm biology. However, more in-depth studies are required to improve the method and develop the applicability of this technique for future experiments.
The authors have nothing to disclose.
Research reported in this publication was supported by Elite Researcher Grant Committee under award number 958680 from the National Institute for Medical Research Development (NIMAD), Tehran, Iran.
12-well culture plates | SPL Life Sciences | 30012 | |
25 cm2 culture flask | SPL Life Sciences | 70325 | |
6-well culture plates | SPL Life Sciences | 30006 | |
Calcium chloride | Sigma-Aldrich | C4901-500G | Working concentration: 2.5 mM |
CMRL 1066 medium | Thermo Fisher Scientific | 11530037 | |
CO2 incubator | memmert | ICO150 | |
D-(+)-Glucose | Sigma-Aldrich | G8270-1KG | |
DMEM | Life Technology | 12100046 | |
Dog bile | Isolated from a euthanized dog and sterilized by 0.2 μm syringe filter | ||
Eosin Y | Sigma-Aldrich | E4009-5G | prepare 0.1% of Eosin for working exclusion test |
Fetal Bovine Serum (FBS) | DNAbiotech | DB9723-100ml | Heat inactivation of FBS (30 min in 40 °C) |
Fetal Bovine Serum (FCS) | DNAbiotech | DB9724-100ml | Heat inactivation of FCS (30 min in 40 °C) |
HEK293T cells | BONbiotech | BN_0012.1.14 | Human embryonic kidney 293T |
HEPES buffered saline (HBS) | Sigma-Aldrich | 51558-50ML | 2x concentrate |
Inverted fluorescence microscope | OLYMPUS | IX51 | |
Penicillin | Sigma-Aldrich | P3032-10MU | Working concentration: 100 IU/mL |
Pepsin | Roche | 10108057001 | Working concentration: 2 mg/mL, pH 2 |
Phosphate-buffered saline (PBS) | DNAbiotech | DB0011 | This reagent solve in less than 1 min in D.W |
Polybrene (Transfection reagent) | Sigma-Aldrich | TR-1003-G | |
RPMI medium | BioIdea | BI-1006-05 | |
Sodium bicarbonate (NaHCO3) | Sigma-Aldrich | S5761-1KG | |
Streptomycin | Sigma-Aldrich | S9137-25G | Working concentration: 100 μg/mL |
Third-generation lentiviral plasmid (pCDH513b) | SBI System Biosciences (BioCat GmbH) | CD513B-1-SBI | Transfer vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran) |
Third-generation lentiviral plasmid (pLPI and pLPII) | Invitrogen (Life Technologies) | K4975-00 | Helper vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran) |
Third-generation lentiviral plasmid (pMD2G) | Addgene | Plasmid 12259 | Helper vector (obtained commercially from Molecular Medicine Research Department of Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad, Iran) |
Tris/EDTA Buffer (TE) | DNAbiotech | DB9713-100ml | |
Trypsin | Sigma-Aldrich | T9935-50MG | 1x working solutions (pH 7.4–7.6) |