Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites

Summary

This protocol describes a methodology to transfect Naegleria gruberi trophozoites with a construct that is maintained throughout passaging trophozoites in vitro, as well as through encystment and excystment.

Abstract

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).

Introduction

The Naegleria genus contains over 45 species, although it is unlikely that all members of the species have been identified¹. Naegleria can exist in different forms: as trophozoites (amoebae), as flagellates, or, when resources are severely limited, as cysts^1,2,3,4. The Naegleria genus is recognized for its one particularly dangerous species, Naegleria fowleri, known as 'the brain-eating amoeba' (reviewed in^{1,2,3,4,5,6,7}), which is the cause of the almost universally fatal primary amoebic meningoencephalitis.

Naegleria encode their rDNA on the CERE located in the nucleolus. Complete sequencing of three Naegleria species' genomes confirms the absence of rDNA in the nuclear genome^8,9,10,11. Based on the limited full-length CERE sequences, the CERE ranges from approximately 10 kbp to 18 kbp in length. CERE of each species of Naegleria carry a single rDNA cistron (containing the 5.8, 18, and 28S ribosomal DNA) on each of approximately 4,000 CERE per cell^12,13,14. Other than rDNA, each CERE has a large non-rDNA sequence (NRS). CERE sizes vary between species; the differences are mainly due to the varying length of the NRS, as the rDNA sequences are highly conserved across the genus^{1,15,16,17,18,19,20}. The mapping of a single origin of DNA replication within the N. gruberi CERE NRS²¹ provides strong support for the hypothesis that Naegleria CERE replicates independently of the nuclear genome.

N. gruberi is a non-pathogenic amoeba often used to study Naegleria biology. We developed a methodology for transforming N. gruberi trophozoites with a CERE clone from the same species to test the hypothesis that Naegleria amoebae tolerate and independently replicate CERE within the trophozoites. This was accomplished by transforming N. gruberi with a full-length clone of N. gruberi CERE into trophozoites and following the fates of the donor CERE clone by polymerase chain reaction (PCR). A general overview of the protocol is outlined in Figure 1. The data presented herein demonstrate that the donor clone can be detected through at least seven passages in tissue culture, as well as be maintained through encystment and excystment. These studies form the basis for a means to dissect how CERE replicates, as well as explore its use as a vector to transfect Naegleria.

Protocol

The details of the species, reagents, and equipment used in this study are listed in the Table of Materials. The sequence of the 17,004 base pair pGRUB construct is provided in Supplementary File 1.

1. Culturing trophozoites

1. Thaw frozen N. gruberi trophozoites at 37 °C for 3 min.
2. Inoculate trophozoites into T25 flasks in modified peptone/yeast extract/nucleic acid/folic acid/hemin with 10% fetal calf serum (PYNFH) media plus 2% buffer (disodium phosphate, potassium dihydrogen phosphate).
3. Culture the trophozoites at 25 °C until ~80% confluent, where they exhibit an amoeboid morphology (Figure 2A).
   NOTE: When initially reviving a culture of trophozoites from their frozen state, it can take up to 5-7 days for a T25 to become confluent. Decant media every 3-4 days and replace with fresh media.
4. Place the confluent flasks on ice for 5-10 min to release the adhered trophozoites from the plastic. Gently swirl the flask to dislodge any remaining adherent cells. The trophozoites now have a 'rounded up' morphology (Figure 2B).
5. Split the cultures 1:2 to 1:4 into new tissue culture flasks.
6. Remove the PYNFH and replace it with sterile encystment media (120 mM sodium chloride, 0.03 mM magnesium chloride, 1 mM disodium phosphate, 1 mM potassium dihydrogen phosphate, 0.03 mM calcium chloride, 0.02 mM iron chloride, pH 6.8)²² to encyst the N. gruberi.
   NOTE: The majority of trophozoites become encysted by 72 h (Figure 2C). Excystment should be completed within 72 h.
7. Remove cysts from flasks, centrifuge at 600 x g for 10 min at 20 °C, and resuspend cysts in PYNFH media with 10% fetal calf serum to excyst the cells. Incubate at 25 °C for 24 h until trophozoites begin to emerge.

2. Transfecting trophozoites

1. Plate the trophozoites into a 6-well flat-bottomed tissue culture plate (1 mL of 1 x 10⁶ trophozoites/mL) and culture at 25 °C.
2. Transfect the cells when they reach 70%-80% confluent (~10.4 x 10⁴ cells/cm² in 6-well clusters). Warm the transfection reagent to room temperature before use.
3. Prepare the plasmid-transfection reagent mixture as follows: 75 ng of plasmid DNA and 0.225 µL of the transfection reagent in 200 µL of PYNFH media without FBS. Prepare sufficient plasmid-transfection reagent mixture to perform transfection in duplicate.
   NOTE: A bacterial plasmid containing a full-length clone of N. gruberi CERE (pGRUB) was used in this study. Empty plasmid (pGEM) was used as a negative control (Figure 3).
4. Incubate the plasmid-transfection reagent mixture at room temperature for 10 min.
5. Remove ~800 µL of medium from the trophozoite monolayers just prior to transfection.
6. Put the plasmid-transfection reagent mixture on the trophozoites.
7. Gently swirl the plate every 15 min to prevent the media from aggregating at the edges of the plate.
8. Wash the monolayers once with 2 mL of growth medium after 1 h incubation at 25 ^°C.
9. Treat the monolayers with 10 U DNase in 1 mL of 10x DNase buffer for 15 min at 37 °C to remove residual plasmid DNA, wash once with growth medium, add 2 mL of fresh media, and place at 25°C. Incubate the plate for 24-36 h.
10. Place the plate on ice for 10 min to release cells.
11. Split one well (1:2) into two new wells.
12. Collect trophozoites from the remaining wells for experimental purposes.
    NOTE: All cultures are performed in duplicate. For each condition, 1 well is used for passage, and 1 is used for CERE isolation.

3. Isolating CERE from Naegleria

1. Calculate the number of trophozoites in each well used for CERE isolation by trypan blue exclusion staining and a hemocytometer²³.
2. Place the trophozoites in a 2.0 mL tube.
3. Wash trophozoites once in 100 mM sodium chloride (pH 7.5) via centrifugation (600 x g, 10 min, 4 °C).
   NOTE: Sodium chloride prevents the trophozoites from adhering to the sides of the tube.
4. Remove the supernatant and resuspend cell pellets in 100 µL of 100 mM EDTA (pH 7.5).
5. Place the tube in a heat block for 15 min at 98 °C to lyse the cells and inactivate the enzymes in the trophozoites.
   NOTE: This step inactivates DNases, which are abundant in Naegleria trophozoites. DNases may interfere with the ability to detect native CERE and the molecular clone in step 4. DNA yield is decreased when over ~3 x 10⁶ cells are used in step 3.4.
6. Centrifuge the tubes at top speed (16,000 x g) at 4 °C for 10 min to pellet the debris.
7. Transfer the supernatants to a new tube.
8. Ethanol precipitate the supernatants with 0.5 volumes of 7.5 M ammonium acetate (pH 7.5), 3 volumes of absolute ethanol, and 1 µL of 10 mg/mL glycogen as a carrier.
9. Invert the tube several times and place the tube at -80 °C for at least 1 h or overnight.
10. Warm the samples to room temperature.
11. Centrifuge the tubes at room temperature for 10 min at 16,000 x g.
12. Remove the supernatant and wash the pellet with 70% ethanol by centrifugation for 5 min at 16,000 x g (at room temperature).
13. Remove ethanol and air-dry the pellet for 5 min.
14. Resuspend the dried pellet in sterile deionized (DI) water. Normalize volume to 10,000 trophozoites per µL.
15. Store at -80 °C until use in PCR.

4. PCR detection of transformed trophozoites

1. Use 20,000 cell equivalents of DNA, 600 nM primers (Table 1), and Taq polymerase and perform the PCR²⁴.
   NOTE: Primers that distinguish between native CERE and the cloned CERE are listed in Table 1, as are the PCR conditions. Figure 3 shows the location of the primers on the constructs. PCR will require optimization depending on the primers used as well as the specific PCR mix utilized.
2. Microwave 0.8% agarose in 1x Tris base, acetic acid, and EDTA (TAE) buffer until the agarose is dissolved.
3. Cool the agarose to ~50 °C at room temperature.
4. Pour the solution into a gel casting tray containing a comb to form wells.
5. Let the gel sit at room temperature until solidified.
6. Place the gel in the electrophoresis apparatus.
7. Decant 1x TAE into the electrophoresis chamber until the gel is covered.
8. Remove the comb.
9. Add 6x loading dye (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol) to samples and load the gel. Use a DNA ladder to determine the size of the PCR product.
10. Attach the electrode, turn on the power supply, and run the gel at 80 V for ~1.5-2 h.
11. Turn off the power and remove the gel.
12. Stain the gel with DNA dye (e.g., 10 µL of 10 mg/mL ethidium bromide) in 100 mL of DI water for 10 min.
13. Destain the gel for 20 min in DI water.
14. Visualize gel on an ultraviolet (UV) light system and document the results.

Results

PCR of trophozoites that have been transfected with pGRUB demonstrates that the transfected CERE is detected through at least seven passages of the trophozoites, as well as encystment and excystment (Figure 4). The primers used in the PCR anneal to both the pGEM vector and the CERE sequence, thereby ensuring that the PCR does not detect native CERE. PCR following transfection of pGEM into trophozoites indicated that pGEM was negative (Figure 4), indicating that ...

Discussion

The protocol outlined herein is very straightforward, although every construct will likely require some degree of optimization, particularly of the DNA-transfection reagent ratio, depending on the nature of the construct and the species of Naegleria used. We have only tested one commercially available transfection reagent using this protocol, but it is likely that several others may be effective. Given that a full-length clone of the CERE is used, containing a functional origin of replication and thus replicatin...

Materials

Name	Company	Catalog Number	Comments
Agarose	Bio Rad	161-3102
Ammonium Acetate	Sigma Aldrich	A-7330
Calcium Chloride	Sigma Aldrich	C-4901
Crushed ice
Culture Flasks, T-75	Thermo Scientific	130190
Culture Plate, 6-well	Corning	3506
DNAse	Sigma Aldrich	D-4527
EDTA, 0.5 M	Affymetrix	15694
Electropheresis Gel Apparatus	Amersham Biosciences	80-6052-45
Eppendorf Tubes, 1.5 mL	Fisher Scientific	05-408-129
Eppendorf Tubes, 2 mL	Fisher Scientific	05-408-138
Ethanol, 100%	Decon Laboratories	2716
Ethidium Bromide	Sigma Aldrich	E-8751
Fetal Bovine Serum	Gibco	26140
Folic Acid	Sigma Aldrich	F7876-25G
GeneRuler 1 kb Plus Ladder	Thermo Scientific	SM1331
Glacial Acetic Acid	Fisher Scientific	UN2789
GoTaq Green PCR Master Mix	Promega	M7122
Heating Block	Thermo Scientific	88871001
Hemacytometer	Hausser Scientific	1483
Hemin	Sigma Aldrich	51280
Iron Chloride	Sigma Aldrich	372870-256
Ligase	NEB	M2200S
Magneisum Chloride	Fisher Scientific	M33-500
Microfuge	Thermo Scientific	MySpin 12
Microscope	Nikon	TMS
N. gruberi	ATCC	30224
Nucleic Acid	Chem Impex Int’l	#01625
Peptone	Gibco	211677
pGEM	Promega	P2251
Potassium Phosphate	Sigma Aldrich	P0662-500G
PowerPac HC Electropharesis Power Supply Unit	Bio Rad	1645052
Sodium Chloride	MCB Reagents	SX0420
Sodium Phosphate, dibasic	Sigma Aldrich	S2554
Tabletop Centrifuge	eppendorf	5415R
Tris, base	Sigma Aldrich	T1503-1KG
Trypan Blue, 0.4%	Gibco	15250-061
ViaFect Reagent	Promega	E4981
Weigh Scale	Denver Instruments	APX-60
Yeast Extract	Gibco	212750