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.
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).
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 cysts1,2,3,4. The Naegleria genus is recognized for its one particularly dangerous species, Naegleria fowleri, known as 'the brain-eating amoeba' (reviewed in1,2,3,4,5,6,7), 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 genome8,9,10,11. 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 cell12,13,14. 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 genus1,15,16,17,18,19,20. 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.
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
2. Transfecting trophozoites
3. Isolating CERE from Naegleria
4. PCR detection of transformed trophozoites
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 the empty plasmid was not retained by the amoebas. Figure 4 demonstrates that pGEM was lost by the first passage – subsequent passages, encystment, and excystment were all negative.
Figure 1: Flow chart of the transfection procedure. Please click here to view a larger version of this figure.
Figure 2: N. gruberi (NEG-M strain) trophozoites in culture. (A) Under standard culture conditions at 25 °C, N. gruberi trophozoites are ameboid in morphology and adherent to the culture flasks/plates. The arrow indicates a pseudopod that is used to propel the trophozoite directionally. (B) Following incubation of the N. gruberi culture on ice, trophozoites become round in morphology and non-adherent. (C) Following incubation in encystment media for 24-48 h, N. gruberi trophozoites become encysted. Scale bars: 10 µm. Please click here to view a larger version of this figure.
Figure 3: pGRUB construct used to transfect N. gruberi (NEG-M). The full-length CERE of N. gruberi (EBM strain, 14,007 bp; red) was cloned into pGEM7Zf(+) (2,997 bp; green), resulting in a construct of 17,004 bp. Arrows on each plasmid indicate the location of the PCR primers. Please click here to view a larger version of this figure.
Figure 4: PCR results detecting transfected pGRUB and pGEM into N. gruberi trophozoites. PCR demonstrates that pGRUB is detected in transfected trophozoites through at least seven passages, encystment (En), and excystment (Ex). pGEM is lost after the first passage of the trophozoites. P1 = passage 1, P7 = passage 7. Negative controls (neg) received no DNA in the PCR, while positive controls (pos) are either pGRUB or pGEM input into the PCR. Expected product sizes are 656 bp (pGRUB) and 337 bp (pGEM). Figures on the left of panel A indicate the size of the brightest bands of the DNA ladder. Please click here to view a larger version of this figure.
Primer Name | Sequence – 5'->3' | Tm | Product Size | |
pGEM-forward | CTCTTCGCTATTACGCCAGC | 54 | 337 bp | |
pGEM-reverse | TTGTGTGGAATTGTGAGCGG | 337 bp | ||
pGRUB-forward | AGCCCCCGATTTAGAGCTTG | 60.5 | 656 bp | |
pGRUB-reverse | TCGGCTAGAAATGAAGTGAGGAC | 656 bp |
Table 1: PCR primers and conditions used in these studies.
Supplementary File 1: The sequence of the 17,004 base pair pGRUB construct. Please click here to download this File.
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 replicating autonomously, the construct possesses all necessary eukaryotic replication machinery. If a partial clone were to be inserted in the pGEM vector, the transfected clone would be predicted to be lost very soon after transfection if it does not contain a functional eukaryotic origin of replication, similar to the empty pGEM vector (Figure 2B). Furthermore, these data demonstrate that the presence of prokaryotic DNA does not interfere with the ability of the molecular clone to be retained by the trophozoite.
While there has been one reported instance of transfection of N. gruberi trophozoites25, the insert used in the present study is considerably larger (~17 kbp) than what was transfected in the prior study (<1 kbp). While it is unknown what the maximum size plasmid can be tolerated by the Naegleria, unpublished data from our laboratory has determined that amebae of another genus (Willaertia) contain CERE that are approximately 24 kbp in size.
A key step in ensuring that this protocol detects DNA within the trophozoite is the DNase treatment step (step 2.9), which ensures that residual DNA (that is, DNA that has not been transfected) is eliminated from the cultures. A couple of quality control procedures were performed to ensure the integrity of the protocol. First, the constructs were sequenced. Second, the PCR products were also sequenced to confirm their identity and ensure that there was no contamination of the assay and confirmed that the PCR products were not the result of mispriming.
While successful transfection of a plasmid with a eukaryotic origin of replication was demonstrated, it is clear from Figure 4 that there is a diminution of the PCR signal following the first passage of the trophozoites. qPCR was performed on the transfected trophozoites over time, and it was determined that the level of pGRUB drops from approximately 1,600 copies in passage 1 to 35 in passage 7 (data not shown), indicating that the transfected plasmid will likely be lost within the next several passages. This is not unexpected, as the transfected plasmid is foreign to the trophozoites and would either be viewed as unnecessary or detrimental to the survival of the amoeba.
There is a fundamental lack of knowledge regarding the CERE of the Naegleria. The majority of CERE sequences in GenBank are partial sequences, with the main focus being on the rDNA and the internal transcribed spacer (ITS) regions, which are used for identifying genus (ITS1) and species (ITS2)1,26,27. While the sequence of the rDNA subunits is highly conserved between Naegleria sp., the NRS of the Naegleria sp. varies greatly, both in size and sequence, ranging from approximately 5.5 kbp to 10 kbp1,15,16,17,18,19,20. The function of the NRS in Naegleria is largely unexplored territory, but it is presumed to contain regulatory elements including, but not limited to, an origin of replication, a promoter, and a termination sequence17. Based on the complete CERE sequences in GenBank, a common feature of Naegleria CERE is the presence of repeat regions in the NRS that are A-T rich. The function of these regions is currently unknown.
The ability to transfect Naegleria trophozoites permits the study of several aspects of CERE NRS biology, particularly the dynamics and requirements of CERE replication. In Escherichia coli, G-quadruplexes have been identified in the promoter regions of some genes28. G-quadruplex structures have also been identified in the promoters of a wide variety of eukaryotes, including mice, rats, humans, and non-human primates29. In silico analysis of several different full-length CERE sequences demonstrates that multiple areas of the NRS are predicted to form G-quadruplex structures. One area of predicted G quadruplex structures in the NRS of several Naegleria sp. is upstream of the beginning of the 18S gene. The ability to transfect trophozoites would permit exploration of identifying the promoter region of the rDNA in the CERE of Naegleria by deleting/mutating sequences upstream of the rDNA. Likewise, similar studies could be performed to identify sequences that terminate rDNA transcription.
The retention of the full-length CERE in the trophozoites for a number of passages will allow for studies that focus on identifying the minimal area of the NRS needed for replication of the CERE. While a region of the CERE has been identified as the putative origin of replication20, it is unknown whether secondary structures outside of the region are required for CERE replication or whether the region could be significantly truncated and the ori remain functional. The repetitive elements in the NRS are found in all Naegleria sp. sequenced to date15,16,17,18,19,20. The purpose of these sequences is currently unknown. Whether they exist as a regulatory element, or if they are deleted, will the CERE replicate in the trophozoites is still unknown. The repetitive sequences are species-specific in Naegleria, but whether the sequences from one species can function in another is not known. In short, there are myriad questions regarding CERE biology that can now be addressed using the technique described herein.
The authors have nothing to disclose.
These studies were partially funded by a grant from the George F. Haddix Fund of Creighton University (KMD). Figure 1 is generated in biorender.com, and Figure 3 is generated in benchling.com.
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 |