Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Summary

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Abstract

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.

Introduction

Cystic echinococcosis (CE) is one of the most important helminth diseases caused by Echinococcus granulosus, a small tapeworm within the family Taeniidae^1,2. 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 disease^3,4,5,6.

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 groups^7,8,9. 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 protozoa^10,11,12.

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 cells^14,15,16. 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.

Protocol

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

1. Collect liver hydatid cysts from naturally infected sheep routinely slaughtered at an abattoir.
2. Completely sterilize the cyst surface using sterile gauze and 70% alcohol before aspirating the protoscoleces (PSCs).
3. Aspirate 20-50 mL of hydatid fluid out into sterile 50 mL conical tubes.
4. After draining, blot out the cyst with a sharp blade, transfer the germinal layer into a sterile 50 mL conical tube, and shake it for a short period (~2 min) to increase the collection of PSCs. Transfer the germinal layer to a new 1.5 mL tube for molecular characterization.
5. Wash the PSCs 3-5x with phosphate-buffered saline (PBS).
6. Observe the PSCs in 0.1% eosin for 5 min to determine the PSC viability under a light microscope. Use PSCs with at least 95% viability for culture.
7. Treat the PSC precipitate with 2 mL of pepsin (2 mg/mL, pH 2) for 15-20 min.
8. To isolate individual PSCs, resuspend the PSC sediment in PBS and pass it through two layers of sterile gauze into a new sterile 50 mL conical tube.
   NOTE: Calculate the average of three counts on 50 µL of PSC sediments using a light microscope. For subsequent trials, use the estimated value to determine PSCs/µL. Each hydatid cyst isolate should be characterized based on nucleotide polymorphisms by molecular methods, such as PCR-sequencing¹⁷ or high-resolution melting curve analysis¹⁸.

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.

1. Prepare the biphasic culture medium to consist of two phases.
   1. For the solid phase, add 10 mL of bovine serum to a 25 mL flask and leave it to coagulate at 76 °C for 20-30 min.
   2. For the liquid phase (modified CMRL), mix 100 mL of heat-inactivated fetal calf serum (FCS), 36 mL of 5% yeast extract, 5.6 mL of 30% glucose (in distilled water), 1.4 mL of 5% dog bile in PBS, 20 mM HEPES buffer, and 10 mM NaHCO₃ in 260 mL of CMRL-1066 medium. Finally, add penicillin (100 IU/mL) and streptomycin (100 mg/mL) to the mixture.
2. Add 10 mL of the liquid phase medium to each 25 cm² culture flask.
3. Add ~5,000 PSCs to the culture flask and transfer the flask to the CO₂ incubator (37 °C, 5% CO₂).
4. After 24-48 h, transfer the PSCs to a new flask with the solid phase (bovine serum) and add 1 mL of the same fresh liquid phase medium per flask. Transfer the flasks into the CO₂ incubator (37 °C, 5% CO₂).
5. Every 5-7 days, replace the medium with fresh liquid phase medium based on changes in the color of the culture medium and the estimated proportion of differentiated PSCs.
   NOTE: Protoscoleces respond quickly to environmental changes after being cultured, and most of them undergo differentiation within 2-3 weeks. Due to the consumption of nutrients and protoscoleces' growth and development, the pH of the culture medium changes, and its color shifts toward orange and yellow.

3. Monophasic cultivation of PSCs of E. granulosus

1. Ensure that monophasic cultivation is done 24-48 h before transduction.
   1. Culture ~5,000 PSCs in a 25 cm² culture flask with RPMI and 10% fetal bovine serum (FBS).
   2. Transfer the flasks into the CO₂ incubator (37 °C, 5% CO₂) until use.

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.

1. Transfer 5 × 10⁵ HEK293T cells to a 25 cm² flask containing 5 mL of DMEM with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin.
2. Incubate the HEK293T cells at 37 °C in 5% CO₂ and passage them in the complete culture medium every 48 h. Finally, use low-passage HEK293T cells for transduction²⁰.

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.

1. Day 1:
   1. After trypsinizing the HEK293T cells²¹ in 6-well culture plates, seed 7 × 10⁴ cells per well with fresh antibiotic-free DMEM containing 10% FBS.
   2. Use cells at 50%-60% confluency for transduction by the calcium phosphate method.
2. Day 2:
   1. Replace the cell culture medium 2-3 h before the experiment with fresh antibiotic-free DMEM containing 2% FBS.
   2. In a 1.5 mL tube, mix the third-generation lentiviral plasmids, including 7 µg/µL pCDH513b (transfer vector), 4 µg/µL PLPII, 4 µg/µL PLPI, and 2 µg/µL PMD2G (helper vectors).
   3. Add HEPES buffer (0.28 M NaCl, 0.05 M HEPES, and 1.5 mM Na₂HPO₄; optimal pH range, 7.00-7.28) to the mixture to a volume of 422 µL.
   4. Add 16 µL of 1% Tris-EDTA (TE) buffer to the mixture.
   5. Add 62 µL of 2.5 mM calcium chloride to the tubes and mix well.
   6. Add 500 µL of 2x HEPES-buffered saline (HBS) to the mixture, drop by drop, within 1-2 min using a Pasteur pipette. Mix gently until a semi-opaque solution is obtained, and incubate the mixture for 20 min at room temperature.
   7. Add the mixture to each plate slowly and distribute it with slow circular motions.
   8. Incubate the plates at 37 °C in a 5% CO₂ incubator and replace the medium after 4-6 h with antibiotic-free DMEM containing 10% FBS.
3. Day 3:
   1. Confirm successful transient transduction and cell viability using an inverted fluorescence microscope.
4. Day 4:
   1. Harvest viruses from the culture medium by collecting each well's supernatant and store them at -70 °C until use.
      NOTE: Harvested lentiviruses could be concentrated and titrated for precise transfection²².

6. Transient transduction of different stages of E. granulosus with the virus

1. Day 1:
   1. Use a 12-well culture plate for gene transfer. Culture 5 × 10³-5 × 10⁴ HEK293T cells in triplicate, with complete DMEM medium as the internal reference.
   2. Culture 150 fresh PSCs in triplicate in RPMI and 10% FBS.
   3. Culture 30 strobilated worms in triplicate in DMEM containing 10% heat-inactivated FBS.
      NOTE: All transduction media must be antibiotic-free. Keep three non-treated control wells for HEK293T cells, PSCs, and strobilated worms in the process.
   4. Prepare a mixture of 1 × 10⁶ viruses with 4 µg/mL transfection reagent (see the Table of Materials) to transduce the HEK293T cells and different stages of the worm.
   5. Add the mixture to each plate slowly and distribute it with slow circular motions. Incubate the plates for 4-6 h in a CO₂ incubator (37 °C, 5% CO₂). Replace the medium for each sample with the same complete culture medium to minimize the toxic effects of the transfection reagent.
2. Day 2:
   1. Repeat steps 6.1.4-6.1.5 to increase the efficiency of transient transduction for the HEK293T cells, PSCs, and strobilated worms.
   2. Incubate all the plates for 24-48 h in a CO₂ incubator with the same culture conditions based on the type of cell media.
3. Day 3:
   1. Check whether the transduction is successful by using fluorescence microscopy.
      NOTE: Consider using further confirmatory assays like PCR/qPCR^20,23 or Western blotting²⁴ techniques in the gene transfer procedure.

Representative Results

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 previously^25,26. 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.

Discussion

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 species^12,27. 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 transduction^12,27. 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 platyhelminths^{8,29,30,31,32}. 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 editing^32,33. 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.

Materials

Name	Company	Catalog Number	Comments
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 Calf 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)