Stable transformation of fluorescent proteins into Nosema bombycis by electroporation

Microsporidia are a group of intracellular parasitic eukaryotes, serious pathogens that cause widespread infection in humans, vertebrates, and invertebrates. Because microsporidia have a thick spore wall structure, the in vitro transformation, cell culture, and genetic operation technology of microsporidia are far behind that of other parasites. In this study, according to an analysis of the life-cycle of microsporidia, Nosema bombycis, and different electro-transformation conditions, the transduction efficiency of introducing foreign genes into N. bombycis was systematically determined. We analyzed the direct electro-transformation of foreign genes into germinating N. bombycis using reporters under the regulation of different characteristic promoters. Furthermore, we systematically determined the efficiency of electro-transformation into N. bombycis under different electro-transformation conditions and different developmental stages through an analysis of the whole life-cycle of N. bombycis. These results revealed that foreign genes could be effectively introduced through a perforation voltage of 100 V pulsed for 15 ms during the period of N. bombycis sporeplasm proliferation. We present an effective method for electro-transformation of a plasmid encoding a fluorescent protein into N. bombycis, which provides new insight for establishing genetic modifications and potential applications in these intracellular parasites.

is undoubtedly the most effective means to improve our understanding of the mechanism of microsporidian infection and proliferation mechanisms.
Since the completion of the detailed genome map of E. cuniculi in 2001, the genome sequences of E. bieneusi, Trichomonas hominis, V. apis, V. ceranae, and N. bombycis have been gradually completed, indicating that the microsporidia studies have entered the era of genome research [18][19][20][21]. The thickness and composition of the spore wall of microsporidia (exospore, endospore, and plasma membrane) complicate the introduction of exogenous genes into microsporidia by currently available technologies, including homologous recombinationbased gene targeting, gene transposition, and CRISPR/ Cas9 gene editing [5,22,23]. At present, the common methods used to explain microsporidia functional genes and the infection mechanism include chemical methods such as fluorescent probe-labeling, biotin-labeling, antibody-labeling, or chemical reagent fluorescent brightener 28 (FB28), and 4′,6-diamidino-2-phenylindole (DAPI) nuclear dye [24][25][26][27]. Furthermore, although it has been reported that RNA interference (RNAi) can inhibit microsporidia gene expression, the interference efficiency is not high, and it is difficult to carry out systematic gene function research [28,29]. Therefore, a method for introducing foreign genes into microsporidia is urgently needed to provide a basis for gene function research, prevention, and control of microsporidia.
Electro-transformation is an effective artificial transformation method that introduces natural or recombinant foreign protein-encoding genes into microorganisms for overexpression or homologous recombination for gene knockout or knock-in [30,31]. It is widely used in the directional transformation of fungi and bacterial strains [32,33]. Since 1993, foreign gene vectors have been introduced by electro-transformation into Toxoplasma gondii for stable and efficient expression, and then into Plasmodium falciparum, Cryptosporidium parvum, and Eimeria intestinalis [34][35][36]. The transformation efficiency of electro-transformation is affected by the growth stage of the parasite, components of the shock buffer, shock voltage, pulse time, electric field strength, resistance, plasmid DNA concentration, components of resuscitation medium, and the resuscitation time [32,37]. At present, there is still no report of the introduction of foreign genes in microsporidia by electric transformation. Therefore, the establishment of such a method is crucial to providing key technical means for analyzing the mechanisms underlying the infection of microsporidia and the interaction between the pathogen and the host.
Microsporidia species have the smallest known eukaryotic genome. Such compactness needs to be investigated and may provide an experimental advantage [20]. Based on the previous experience of successful in vivo transformation in parasites, when designing a successful in vivo gene transformation method of N. bombycis, we first analyzed the efficiency of fluorescent protein expression by the original Bombyx mori A3 cytoplasmic actin promoter (A3) and Orgyia pseudotsugata multiple nucleocapsid nuclear polyhedrosis virus immediate-early 2 gene (OpIE2) promoter. Then, the N. bombycis transduction efficiency was explored according to the previously reported electro-transformation strategy. Finally, based on the analysis of the life history of N. bombycis, we discuss the best conditions for the electro-transformation of foreign genes into N. bombycis. The successful introduction of a fluorescent protein gene by electro-transformation undoubtedly allows foreign gene recombination, gene fusion expression, nonhomologous end joining, and gene editing in the N. bombycis genome.

Vector construction
The plasmid pBac-A3 prm -enhanced green fluorescent protein (EGFP) and pIZ-OpIE2 prm -Discosoma sp. red fluorescent protein (DsRed) was constructed by our laboratory. Briefly, the DsRed fragments were amplified using the primers DsRed-F: CCC AAG CTT ATG GCC TCC TCC GAG AACGT and DsRed-R: CGG GGT ACC CAG GAA CAG GTG GTG GCGG. Subsequently, the polymerase chain reaction (PCR) fragments were connected to a pIZ-V5/His vector to generate pIZ-OpIE2 prm -DsRed. The plasmids pBac-A3 prm -EGFP were constructed using the B. mori cytoplasmic actin gene BmA3 promoter and the enhanced green fluorescent protein (EGFP) sequence, in addition to the SV40 polyadenylation signal sequence as described previously [40].

Immunofluorescence
The microsporidia localization at different infection times was analyzed by immunofluorescence after infection of BmN-SWU1 and BmE-SWU1 cells with N. bombycis. Briefly, the sample was washed with phosphate-buffered saline (PBS), and 500 µl of 4% paraformaldehyde was then added to each well and left at room temperature for 15 min. After washing with PBS, 500 µl of 1% Triton X-100 was added and the sample was left at room temperature for 10 min, then incubated at 37 °C for 1 h. Finally, the samples were incubated with anti-PTP2 antibody (1:200, rabbit), incubated at 37 °C for 1 h, and then incubated with Alexa 488-conjugated goat antirabbit secondary antibody (1:1000) at 37 °C for 1 h. The nuclei-stained microsporidia were incubated with FB28 at 37 °C for 1 h. An Olympus FV3000 scanning electron microscope was used for image acquisition. Excitation wavelengths of 405, 488, and 561 nm were used for all the images.

Electroporation of N. bombycis
Four hundred microliters of 1 M sorbitol and 5 µg of plasmid were added to a solution containing 1 × 10 7 spores, then resuspended and mixed evenly, precooled on ice for 10 min, transferred to a precooled electrode cup, and left there for 5 min. The electric shock parameters were set, the shock was applied, the sample was left standing for 3 min and then washed, and BmN-SWU1 and BmE-SWU1 cells were then infected after 0.1 M KOH treatment. The electroporation system (CUY21EDIT II) was purchased from BEX Japan.

Electroporation of cells
The BmN-SWU1 and BmE-SWU1 cells were collected in a 5 ml centrifuge tube and centrifuged at 800×g for 5 min, and the supernatant was removed. Next, 3 ml Opti-MEM (minimal essential medium) was added, the cells were centrifuged for 800×g for 5 min, and the supernatant was removed. Then, 25 µl of medium (cell content ≥ 1 × 10 6 ) and 5 µg of plasmid were added, and the mix was transferred to a 2 mm electrode cup. Finally, the electro-transformation parameters were set, and the cells were electroporated and left to recover for 1 min. After electroporation, 200 µl of Opti-MEM medium were added to the electrode cup, and the cell suspension was transferred to 24-well plates. The cells were cultured at 27 °C, and the expression of the transformation plasmid was observed for 24 h.

Statistical analysis
All of the statistical analysis was performed using Student's t-test in GraphPad Prism 6 (http:// www. graph pad. com) (GraphPad Software, Inc., San Diego, CA, USA). All the data are presented as the mean ± SD. Differences between groups were considered significant at the following significance levels, **P < 0.01.

Plasmid transfection into N. bombycis-infected cells
To analyze the expression of fluorescent protein after BmE-SWU1 cell infection with microsporidia, the B. mori actin 3 (A3) promoter and baculovirus Orgyia pseudotsugata multi nucleocapsid nuclear polyhedrosis virus (OpMNPV) OpIE2 promoter were selected to construct fluorescent protein expression vectors (Fig. 1a).
Since the best time for introducing a foreign gene into N. bombycis is unclear, multiple transfection at different times was performed to observe fluorescent pro- was clearly observable after transfection with pBac-A3 prm -EGFP and pIZ-OpIE2 prm -DsRed of N. bombycisinfected cells. In addition, the A3 and OpIE2 promoter expression vector could enter N. bombycis and express the fluorescent protein (Fig. 1b).

Electro-transformation of fluorescent protein into N. bombycis spores
To obtain high fluorescence intensity trace-N. bombycis, we attempted to electro-transform N. bombycis with a fluorescent protein-encoding plasmid. We analyzed the germination of N. bombycis with different electrotransformation parameters. Different electro-transformation conditions had some effects on the germination of N. bombycis, but all of them could normally germinate (Fig. 2a). Notably, the germination rate of N. bombycis was relatively high under the condition of 2.5 kV, 200 Ω, and 25 µF electric transformation (Table 1). Therefore, BmN-SWU1 cells were infected with N. bombycis under this electric-transformation condition (Fig. 2b). However, the expression of fluorescent protein could not be detected in N. bombycis. These results showed that the electro-transformation of N. bombycis could not introduce the plasmid into N. bombycis.

Tolerance of BmN-SWU1 and BmE-SWU1 cells to electro-transformation
Electro-transformation can electroporate the cell membrane, allowing the plasmid to enter the cell membrane instantaneously [33,41]. Therefore, we carried out an electro-transformation experiment of microsporidia at the cellular level.  length 60 ms, pulse interval 50 ms, and 10 cycles were the best electro-transformation conditions in perforation electrophoresis ( Table 2).

Analysis of N. bombycis life history in BmN-SWU1 cells
To determine the best electro-transformation period, we labeled the polar tube protein NbPTP2 and nuclear state during N. bombycis infection by immunofluorescence. Nosema bombycis ejected polar filaments to infect host cells, and NbPTP2 could be labeled after 3 h p.i. At 9-21 h p.i., the period of schizont N. bombycis with obvious membranc lysis could be observed (Fig. 3). At 48-72 h p.i., the form of binuclear and polynuclear of N. bombyxcis were stained by DAPI. There was no sporoblast during at this time. It is speculated that the sporoblast began to develop 48-72 h p.i. At 96 h p.i., the spores entered host cells by ejecting polar filaments (Fig. 3). These results showed that 3-72 h p.i. is the critical period for N. bombycis proliferation, and an important period of electrical transformation.

Transfection of N. bombycis for stable expression of fluorescent protein
Based on the life history of N. bombycis infections, we selected 9 h, 21 h, and 48 h after infection for the electro-transformation experiment. We introduced pBac-A3 prm -EGFP and pIZ-OpIE2 prm -DsRed plasmids into the BmE-SWU1 cell line by electro-transformation. The green fluorescence proteins could be detected at 9 h p.i., 21 h p.i., and 48 h p.i., which indicates that the best period for foreign gene introduction is in the sporoplasm and sporogony stage of N. bombycis ( Fig. 4a and b). It is speculated that N. bombycis has only one plasma membrane in the intracellular stage at 9-48 h p.i. Therefore, the foreign gene could easily enter N. bombycis by electro-transformation. Fluorescence observation also showed that regardless of the period of electro-transformation, the fluorescence signal could be detected at 84 h  The results showed that the fluorescence signal of several spores could be detected in a single image (Fig. 4c and d).

Discussion
Microsporidia, a large class of unicellular eukaryotes with typical obligate intracellular parasitism, have been identified in more than 200 genera and 1500 species [42]. However, up to now, there have been no reports of gene mutation, genome deletion, or fusion expression of foreign genes in microsporidia, which seriously affects the interpretation of the microsporidian development cycle and the analysis of the infection mechanism. To overcome these challenges, in this study we successfully introduced genes encoding foreign fluorescent proteins into microsporidia by electro-transformation, and systematically analyzed the electro-transformation conditions and best opportunity for foreign gene introduction.
Microsporidia are unique intracellular parasites. The introduction of foreign genes into infected BmN-SWU1 through non-transposon vectors and the attempt to culture N. bombycis in vitro could not efficiently introduce foreign genes into the parasite [14,43]. As an important transformation technology in molecular biology, electrotransformation has the advantages of a wide application range, high transformation efficiency, and simple operation [31][32][33]. It can be applied to microbial genetic engineering, mutation breeding, exogenous expression of related proteins, and directional transformation of strains [37,39]. Therefore, this study attempted to introduce foreign genes into microsporidia through electro-transformation. We first tried direct electro-transformation of the N. bombycis, but no stable expression of foreign genes was reached. Given what is known about electrical transformation in Plasmodium and Cryptosporidium, we speculate that failure to transform the germinated microsporidia may be because, during this period, the spore protoplasm is not completely exposed to the culture medium, and the culture medium is not suitable for N. bombycis (Fig. 2) [35,44,45]. Exploring the electro-transformation methods in N. bombycis and the culture conditions is suitable for the proliferation of N. bombycis [45]. Therefore, we further analyzed the intracellular electrotransformation at different stages after infecting cells with N. bombycis and determined that the schizogony of N. bombycis was the key period for electro-transformation (Fig. 3). These results verified the electro-transformation conditions of microsporidia, which proved that changing the permeability level of the membrane could transform foreign genes more effectively and confirmed that foreign genes could not be effectively introduced during spore germination and mature spore morphology (Fig. 5). Expression of fluorescent marker genes, homologous repair, gene design, anti-drug screening, gene knockout, and CRISPR library screening have been widely used in Toxoplasma gondii after transient transformation and expression of foreign genes based on electrotransformation [34,[46][47][48]. Therefore, based on the electro-transformation method, we can carry out further studies in microsporidia, as described in the following three examples: (1) According to the previous studies on methionine amino-peptidase (MetAP2)resistant fumagillin, we could express fluorescent protein fusion resistance markers to screen the strains stably expressing the fluorescent proteins [45,49]. (2) A long terminal direct repeat retrotransposon (LTR) transposon and a Tc1-like DNA transposon have been reported in the N. bombycis genome, and PiggyBac transposon could also be transposed in the genome of P. falciparum [45,[50][51][52]. Therefore, we could combine fluorescent protein, resistance marker, and transposon to transform foreign genes into the N. bombycis genome through electrical transformation. (3) CRISPR/ Cas9 genome editing techniques are a powerful tool developed for many intracellular parasites. It is not difficult to imagine that genetic manipulation mediated by CRISPR will also be applied to the genome of microsporidia parasites as these techniques improve [46,47]. It was previously reported that after inducing spore germination in vitro, the sporoplasm of N. bombycis was successfully isolated and identified [53,54]. Combined with electro-transformation technology, it may be more efficient to introduce foreign genes in N. bombycis, which will enable rapid development in the genetic operation of N. bombycis.

Conclusions
In conclusion, we systematically analyzed a method for successfully transforming exogenous genes into N. bombycis. In this study, we determined the conditions of punctured voltage, pulse length, and pulse interval, and we found that the schizogony period of N. bombycis is the best for a successful transformation. These results provide important insights for the development and application of N. bombycis genetic modification techniques.