Fast and efficient molecule delivery into Euglena gracilis mediated by cell‐penetrating peptide or dimethyl sulfoxide

This study describes the development of two methods for delivering exogenous materials into Euglena gracilis, a unicellular flagellate organism. We report that the use of Pep‐1, a short cell‐penetrating peptide (CPP), or dimethyl sulfoxide (DMSO) can mediate fast and efficient intracellular delivery of exogenous materials into E. gracilis, achieving cellular entry efficiency as high as 70–80%. However, compared with human cells, the penetration of this algal cell with CPP requires a much higher concentration of purified proteins. In addition, upon convenient treatment with DMSO, E. gracilis cells can efficiently adsorb exogenous proteins and DNA with 10% DMSO as the optimal concentration for Euglena cells. Our results provide more options for the E. gracilis transformation ‘toolkit box’ and will facilitate future molecular manipulations of this microalgal organism.

methods based on CPP and DMSO utilizing their ability to penetrate or disturb cell membranes. Our results suggested that both methods applied to E. gracilis cells and achieved fast and efficient delivery results.

Chemicals and microorganisms
Euglena gracilis strain FACHB-848 was ordered from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (Wuhan, China) and cultured according to its instructions in the HUT medium [10]. DMSO was ordered from MP Biomedicals and trypan blue from TargetMol. PCR PCR reactions were conducted with Phanta Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). PCR primers used in this work, as listed in Table 1, were synthesized from Tsingke. To check the presence of pPZP211 plasmid in DMSO-treated E. gracilis cells, DNA was extracted from alkaline lysed cells and used for PCR with primers 211-L5sc_F and mGFP6seq_R. For amplifying the Prp8 fragment, primers (egP8_F/Cy5-egP8_F/FITC-egP8_F and egP8_R) were designed according to the E. gracilis Prp8 nucleotide sequence (Genbank accession number OP185589) that was previously assembled. After extracting total RNAs from E. gracilis cells with Trizol, reverse transcription was performed with FastKing RT Kit With gDNase (TIANGEN, Beijing, China). To visualize the PCR fragment in the cell, the forward primers (Cy5-egP8_F/FITC-egP8_F) were additionally labeled with a Cy5 or FITC dye at their 5 0 termini synthesized also by Tsingke (Beijing, China).

Plasmid construction
Plasmids used here were constructed with a ligationindependent cloning method (ClonExpress II One Step Cloning Kit, Vazyme) and based on the pET21-EGFP plasmid, which contains an EGFP coding sequence flanking with an upstream T7 tag and downstream 69 His residues (Fig. 1). pET21-PGH was constructed by replacing the T7 tag sequence (MASMTGGQQMG) on pET21-EGFP with Pep-1 (LETWWETWWTEWSQPKKKRKV), gene synthesized from Tsingke Biotechnology and amplified with primers (21G-pep-1sc_F and 21G-pep-1sc_R) except the first Methionine [11,12]. pET21-GPH was constructed by inserting the Pep-1 fragment (amplified with GP_oF and P21sc_R) into pET21-EGFP. For constructing pET21-HGP, the GFP-Pep-1 fragment was amplified from pET21-GPH with the forward primer 21HGPsc_F containing the 69 His sequence and the reverse primer 21HGPsc_R containing a stop codon after the Pep-1 sequence. Subsequently, the amplified GFP-Pep-1 fragment was inserted into the BamHI/XhoI-linearized pET21-EGFP to generate pET21-HGP. Similar to pET21-HGP, pET21a-HPG was constructed by amplifying the Pep-1-GFP fragment from pET21-PGH with the forward primer 21HPGsc_F containing the 69 His sequence and the reverse primer 21HPGsc_R containing a stop codon after the EGFP sequence, and then inserted into the NdeI/XhoI-linearized pET21-EGFP.

Protein purification and SDS/PAGE
Plasmids were transformed into BL21 (DE3) and expressed upon adding IPTG for 3 h at 37°C. Subsequently, cells were harvested by centrifugation, dissolved in buffer A (20 mM Tris-HCl, pH 8.0, and 25 mM NaCl) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and then broken by sonification. Soluble His-tagged Pep-1 fusion proteins were affinity purified with Ni-NTA resin. Thirty millimolar imidazole was used to remove unbound proteins from the resin, and 300 mM to elute Pep-1 proteins. The eluted proteins were further concentrated by ultrafiltration and dialyzed against phosphate buffer saline (PBS). Purified proteins were applied to 10% SDS/PAGE and visualized with coomassie staining. For the delivery experiment, proteins were diluted to different working concentrations with an HUT medium.

Intracellular delivery experiment
Euglena gracilis cells were collected by centrifuging at 1000 g for 1 min and washed once with flesh HUT medium. Subsequently, cells were added with prepared proteins of different concentrations to a final cell density of 1 O.D (0.86 9 10 6 cellsÁmL À1 ). Cells were then incubated at 37°C (for CPP) or 25°C (for DMSO) at different times. For DMSO treatment, cells were incubated with an HUT medium containing different concentrations of DMSO for 1 h, then washed with PBS three times before adding exogenous proteins or nucleic acids.

Cell viability assay
After treatments of electroporation, PGH protein or DMSO, cells were stained with 4 mM trypan blue for 3 min, centrifuged for 1 min at 1000 g, washed with PBS, and then observed under a microscope.

Microscopy
Euglena gracilis cells were collected, washed with PBS several times, and observed under the fluorescence microscope BX63 (Olympus) at 209 or 1009 magnification. For DAPI staining, cells were fixed with 4% paraformaldehyde for 20 min, treated with 0.5% Triton X-100 for 15 min, and stained with 1 lgÁmL À1 DAPI for 5 min before observation.

Statistical analyses
Experiments for both Pep-1 and DMSO delivery were repeated at least three times and statistical analyses were performed with PRISM7 (GraphPad, Boston, MA, USA). Using a significance level set at 95%. Based on statistically significant differences, the Tukey's multiple post-test (for PGH delivery) and the one-way analysis of variance test (for DMSO delivery) were used to analyze the data of each group. For PGH and DMSO delivery, the difference was considered statistically significant when P < 0.0001 and P < 0.05, respectively.

Purification of Pep1 fusion GFP proteins
To investigate CPPs-mediated delivery of exogenous materials into E. gracilis, the 21-amino acids amphipathic CPP peptide Pep-1 was selected for our research [11]. The nucleic acids sequence of Pep-1 was synthesized according to Choi et al. [12]. Plasmid pET21-EGFP, which contained an EGFP coding sequence, was used as a template for constructing Pep-1-related plasmids as mentioned in Materials and methods ( Fig. 1). Pep-1 was inserted into pET21-EGFP between the T7 tag and EGFP to obtain the plasmid pET21-PGH (Figs 1 and 2A). Subsequently, this plasmid was transformed in BL21(DE3) strain for expression of the 31-kD PGH protein and purification via Ni-NTA affinity chromatography (Fig. 2B).
Besides the pET21-PGH plasmid, three other Pep-1-related plasmids were constructed for the purpose mentioned below. These three plasmids, pET21-HPG, pET21-HGP, and pET21-PGH, differed only in the positions of His tag and Pep-1 (Figs 1 and 2A). Under the same experimental conditions as for pET21-PGH, these plasmids were successfully expressed, and the corresponding proteins with similar sizes were expressed and purified (Fig. 2B). Moreover, a pET21-GPH plasmid without the T7 tag sequence was constructed. However, we did not detect the protein expression of this plasmid after IPTG induction, suggesting that the T7 tag sequence is required for GPH expression, agreeing with a previous report [13].

Intracellular delivery of PGH into E. gracilis cells
Pep-1 fusion protein could enter cells as low as 0.25 lM and as short as 15 min [12]. However, when 5 lM of PGH was added to E. gracilis cells, no fluorescence signal was observed after incubation at 25°C for 12 h. Only after increasing the concentration of PGH to 50 lM could we observe very few fluorescence signals (Fig. 3A). Because human cells are usually cultured at 37°C, we speculated PGH delivery might be enhanced in E. gracilis cells at this higher temperature. Indeed, much more fluorescence signals were found at 37°C than at 25°C (compare Fig. 3B to Fig. 3A). Accordingly, we conducted Pep-1 fusion protein delivery at this higher temperature hereinafter. Three concentrations of PGH for E. gracilis delivery were tested (Fig. 3B). When 5 lM of PGH was added to E. gracilis cells, no fluorescence signal was observed after 2 and 7 h of incubation. Even after 12 h of incubation, only very few cells (~1%) exhibit the fluorescence signals (Fig. 3B,C). Consequently, the PGH concentration was added to 50 and 100 lM. Our data indicated more than 25% and 60% delivery efficiencies at 50 and 100 lM of PGH after 7 h of incubation, respectively (Fig. 3B,C). Longer incubation up to 12 h only slightly increased the delivery efficiency (70% for 100 lM). We noticed that the transduced PGH proteins were evenly distributed in the cell except some dark areas overlapped with chloroplasts (1009 magnification image in Fig. 3B). Although the PGH concentration added to E. gracilis cells was much higher than that to human cells, our results showed that PGH-transduced E. gracilis cells remained motile but less active than they were at physiological temperature. The viability of PGHtransduced cells was also confirmed by staining with trypan blue (Fig. 5). Compared with electroporated cells, only a few PGH-treated cells (~5%) were stained blue. For all these delivery conditions, our results detected no fluorescence signal with the Pep-1free GFP protein (Fig. 3B).

Position of Pep-1 influenced delivery efficiency
Besides the above PGH protein, three other Pep-1related plasmids were constructed and respective proteins were purified (Fig. 2). As mentioned above, these proteins only differ in Pep-1 and His tag positions. The purpose of the purification of these proteins was to investigate the influence of Pep-1 position on delivery efficiency. Indeed, upon adding these three proteins, together with the PGH mentioned above, to E. gracilis cells, our findings suggested that delivery efficiencies of PGH and HPG were much higher than those of HGP and GPH (Fig. 3D). This result suggested that N-terminal Pep-1 promoted intracellular delivery more efficiently, consistent with a previous report [14].

Intracellular delivery mediated by DMSO
As mentioned above, a higher temperature (37°C vs. 25°C) promoted the delivery of PGH. Because higher temperatures increase the fluidity of cellular membranes, we speculated reagents that affect membrane fluidity might also influence the delivery of exogenous materials. Here, this idea was tested with DMSO, a commonly used laboratory reagent that can enhance membrane permeability. E. gracilis cells were treated with different concentrations of DMSO for 1 h. After removing DMSO by washing with PBS, cells were added with the GFP protein without the Pep-1 tag and incubated for 12 h. The permeation of GFP in E. gracilis cells also exhibited a concentrationdependent manner of DMSO, indicative of a similar effect as Pep-1 (Fig. 4A,B). With 10% DMSO,~80% delivery efficiency was observed with high viability, as confirmed by trypan blue staining (Fig. 5).
Besides the above delivery with proteins, DNAs were also examined. A pair of primers (Table 1) was designed to amplify a 1.7-kb fragment of the E. gracilis Prp8. To directly observe the intracellular localization of this DNA fragment just as that of the above fluorescent protein, a fluorescent dye Cy5 was conjugated with the 5 0 of the forward primer. With the same delivery procedure as GFP, the red fluorescent signals of this DNA fragment in E. gracilis cells were also observed under the microscope (Fig. 4C). Notably, the intracellular delivery efficiency of this DNA fragment was even higher than that of the above GFP protein. Additionally, to quench any fluorescent signals attached to the cell surface, cells were stained with trypan blue after being treated with DMSO and FITC-labeled. Our findings revealed that the cellular localization of the Prp8 fragment displayed the same patterns as above (Fig. 4D).
Finally, to examine the feasibility of plasmid delivery into E. gracilis cells with DMSO, a plasmid of 12 kb in size bearing the pPZP211 backbone was used for this purpose. Upon DMSO treatment for 1 h and removal by washing with PBS, the plasmid was added to E. gracilis cells at a final concentration of 10 ngÁlL À1 . After 3 days of culture, cells were washed, and cellular DNAs were extracted. A 736-bp fragment was detected from DMSO-treated cells (Fig. 4E). In comparison, no DNA was amplified from cells without DMSO treatment. This result suggested that a plasmid as large as 12 kb can enter E. gracilis cells with the help of DMSO.

Discussion
As an evolutionarily unique and model organism, E. gracilis has been studied for, from scientific questions to biotechnology solutions. All these studies on E. gracilis demand more methodologies. We here developed CPP-and DMSO-mediated transduction methods for delivering exogenous materials into this algal cell. Compared with available methods, such as Agrobacterium-mediated nuclear transformation, electroporation, and microinjection, the methods described here are fast, convenient, and efficient.
Under our experimental conditions, the delivery of Pep-1 conjugated PGH to E. gracilis cells resulted in approximately 70% efficiency. Notably, E. gracilis requires a much high concentration for cell entry than human cells. Besides Pep-1, an arginine-rich cationic CPP peptide TAT (GRKKRRQRRR) was tested, and the results indicated delivery concentration and efficiency of TAT-EGFP are comparable to PGH. A unique microtubular structure underneath the cell membrane in E. gracilis cells is termed the pellicle [15]. We speculated the pellicle might hinder the entry of CPP, and only a higher concentration of CPPs can break through this barrier. It will be interesting to check whether the CPP delivery efficiency can be increased to that of humans after disrupting the pellicle. Because the molecular composition of the pellicle is so far unclear, this hypothesis cannot be examined here [16]. Although it appears E. gracilis cells are refractory to CPP delivery, further investigation and optimization will make this delivery peptide more applicable.
For DMSO delivery,~80% of E. gracilis cells displayed the fluorescent signals of exogenous GFP protein and Cy5-labeled DNA fragment with 10% DMSO. Also, higher concentrations of DMSO were tested. Although these treated cells showed a relatively higher delivery efficiency, they were more inactive than 10% DMSO-treated cells. In addition, a previous study in human cells found that DMSO aided the penetration of GFP with a CPP peptide but failed to do so without the peptide [9]. By contrast, our study showed that DMSO is effective for E. gracilis cell delivery even without CPP. Therefore, DMSO is applicable as an independent delivery method in E. gracilis cells, similar to its role in yeast transformation [8].
Usually, delivery of RNA or large ribonucleoprotein complexes in E. gracilis depends on traditional electroporation [17,18]. Although our methods tested only protein and DNA molecules here in E. gracilis, they should be suitable for delivering other exogenous materials. In addition, our methods may be applied to other algals as well.