- Email: [email protected]
Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus
PARINT-01488; No of Pages 4 Parasitology International xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus Hirokazu Sakamoto a,1, Kiyoshi Kita a,b, Motomichi Matsuzaki a,⁎ a b
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, Japan
a r t i c l e
i n f o
Article history: Received 31 December 2015 Received in revised form 15 March 2016 Accepted 5 April 2016 Available online xxxx Keywords: Perkinsus marinus Transfection Drug selection Bleomycin
a b s t r a c t Perkinsus species are notorious unicellular marine parasites that infect commercially important mollusk species including clams and oysters. Recent accumulation of molecular information will greatly facilitate the understanding of Perkinsus biology and development of strategies to control infection. However, the limited availability of methods for genetic manipulation has hindered molecular-based studies of the parasites. In particular, the lack of a drug selection system requires manual isolation of fluorescent cells under a microscope to establish transfected cell lines. Here, we introduce a drug selection system using a glycopeptide antibiotic, bleomycin, and a vector containing the resistance gene Sh-ble. Perkinsus marinus is sensitive to bleomycin, and 100 μg/ml of this drug completely blocks its proliferation. Concomitant expression of Sh-ble enables us to specifically select transfected cells in the presence of the drug. We believe that this system provides new opportunities for functional analyses of this parasite. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Background
2. Objective
Perkinsus is a unicellular marine eukaryote and a member of the Perkinsozoa, a recently recognized group of parasites sister to dinoflagellates [1]. They are notorious for infecting commercially important bivalve species like clams and oysters worldwide [2]. For example, P. marinus causes Dermo disease in the wild and cultivated eastern oyster, Crassostrea viginica, in the eastern coasts of the United States. Perkinsus olseni infects a wide variety of clam species including the Manila clam, Ruditapes philippinarum, and is widespread throughout the tropical and temperate Pacific Ocean as well as the Mediterranean Sea. Methods of controlling diseases caused by these parasites are urgently needed. Beyond the needs of fisheries, Perkinsus spp. are uniquely important in evolutionary biology because they may share a phototroph ancestor with apicomplexan parasites and have independently lost their photosynthetic ability [3]. Comparative analyses between these parasites, especially of the chloroplast remnants, will facilitate understanding of how photosynthetic organisms become parasites. Although a draft genome sequence of P. marinus (DDBJ/ENA/GenBank accession AAXJ01000000) is valuable for this purpose, experimental studies using this molecular information are quite limited, probably because only a small set of genetic manipulation techniques is available [4].
Transgenic techniques are a convincing approach used for functional analysis of proteins of interest. These techniques are currently used in numerous studies of apicomplexan parasites including Plasmodium spp. and Toxoplasma gondii. In P. marinus, however, a drug selection system has not been established, although feasibility of transfection has been reported [4]. If a stably transformed line is required, a small number of cells expressing the transfected gene fused with green fluorescent protein (GFP) must be manually isolated from untransfected cells under a fluorescence microscope. Here, we will introduce a handy assay for parasite growth to screen selection drugs and a selection system using bleomycin (ble) and its resistance gene.
⁎ Corresponding author at: Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail address: [email protected] (M. Matsuzaki). 1 Present address: Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan.
3. Methods 3.1. Parasites Many strains of Perkinsus spp. are available from American Type Culture Collection (ATCC). We use P. marinus strain CRTW-3HE (ATCC 50439) and maintain it at 26 °C in ATCC medium 1886. Lipid mixture (1000×) (Sigma-Aldrich, Saint Louis, MO) can be used as a substitute for a 100× lipid concentrate indicated in the ATCC's instructions. Instant Ocean Sea Salt (Aquarium Systems, Sarrebourg, France) and Daigo's Artificial Seawater SP (Wako Pure Chemical Industries, Osaka, Japan) can be used as synthetic sea salt. Subculture 1 ml of stationary-phase culture in 10 ml of medium in a T-25 culture flask once per week.
http://dx.doi.org/10.1016/j.parint.2016.04.003 1383-5769/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: H. Sakamoto, et al., Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.04.003
2
H. Sakamoto et al. / Parasitology International xxx (2016) xxx–xxx
by site-directed mutagenesis. The coding sequence of PmMOE was replaced with multifunctional GFP (mfGFP) [5] and then mCherry. The bleomycin resistance gene Sh-ble was amplified from the pLEXSY_Ible2 plasmid (Jana Bioscience, Jena, Germany) and inserted as a downstream fusion of mCherry. As a result, the translation product will be mCherry fused with downstream Sh-ble containing an additional Nterminal peptides derived from a multi-cloning site of pColdIII vector (Fig. 1). For transfection, prepare plasmids in more than 1 μg/μl. Fig. 1. A schematic diagram of the transfection plasmid pMOE-mCherry-ble.
3.2. Drug preparation Dissolve bleomycin (Wako Pure Chemical Industries) in water at 10 mg/ml. Filter the solution with an 0.22-μm Ultrafree-MC centrifugal filter unit (Merck Millipore, Billerica, MA) and store the small aliquots at −80 °C until use. 3.3. Drug response assay Dispense 80 μl of culture medium containing 1.0 × 105 log-phase trophozoites per well in a 96-well flat-bottom microtiter plate. Add 20 μl of various concentrations of bleomycin or other drugs to be tested, and incubate the plate at 26 °C for 3 days. Add 10 μl of Cell Counting Kit8 solution (Dojindo Laboratories, Kumamoto, Japan) to each well, and incubate the plate at 26 °C for 1.5 h. Measure the absorbance at 450 nm with a reference wavelength at 600 nm using a microplate reader. We use a SpectraMax M2e-TUY microplate reader (Molecular Devices, LLC, Sunnyvale, CA). Seal the test wells with a sterilized platesealing film to prevent the medium from evaporating during the incubation. On day 0, prepare additional triplicate wells containing the same density of log-phase trophozoites without drug, and measure their absorbance after treatment with Cell Counting Kit-8. Calculate the relative growth as follows: (OD450–600 of culture with drug − OD450–600 at day 0)/(OD450–600 of culture without drug − OD450–600 at day 0) × 100 [%], where OD450–600 means the optical density at 450 nm subtracted with the optical density at 600 nm. Always make triplicates and perform three independent assays. 3.4. Plasmid for transfection The plasmid pMOE-mCherry-ble is available upon request. Construction of this plasmid was performed as follows. We first constructed the transfection plasmid pPmMOE as described in the original report [4]. The two BspHI sites in the backbone were converted to PciI sites
3.5. Transfection and selection of transfectants Harvest 5 × 107 log-phase trophozoites from day 2 or day 3 culture by centrifugation at 800×g for 5 min at 26 °C. Resuspend the cell pellet in 100 μl of Solution 2 supplemented with P2 included in the Basic Parasite Nucleofector Kit 2 (Lonza, Basel, Switzerland). Add 5 μg of supercoiled plasmid, and then perform electroporation using the D023 program in Amaxa Nucleofector II (Lonza) [4]. Dilute the cells with 2 ml of culture medium, dispense them into two wells in a 6well plate, and incubate at 26 °C for several days. Confirm transiently expressed mCherry signal, and then dilute 50 μl of the culture in 1 ml of the medium containing 100 μg/ml of bleomycin in another well. Tightly seal the plate with a sterilized plate-sealing film and incubate for up to two months without changing the medium. 3.6. Flow cytometry analysis (optional) Dilute P. marinus cells to less than 1 × 106 cells/ml in 1 ml of ATCC medium 1886 without lipid mixture, JLP carbohydrate, and fetal bovine serum. Analyze the cells with a flow cytometer after passing cell suspension through a 35-μm nylon mesh cell strainer (Becton Dickinson, Franklin Lakes, NJ). 4. Special remarks/comments 4.1. The drug response assay system This assay system is similar to the method previously reported for P. marinus [6], although the tetrazolium salt used is different. Cell Counting Kit-8 requires optimization of the incubation period for cell number determination. The 1.5-h incubation gives us the best correlation between absorbance and cell number (Fig. 2A), but it may depend on species, strain, culture medium, and temperature. The linearity range was 1 × 106 to 1 × 107 cells/well (R2 = 0.9995) in our experimental setup (Fig. 2B). Seeding density is critical (Fig. 2C) because the growth rate of P. marinus depends on an autocrine factor and therefore cell density [7]. We used a seeding density of 1 × 105 cells/well and a culture time of 3 days.
Fig. 2. Effect of parameters on the cell viability assay. (A) Effect of incubation time on the correlation between cell numbers and the optical density (OD) values. The incubation times (h) are shown in the graph. (B) Regression between the OD value and cell number within the 1 × 106 to 1 × 107 cells/well range when the WST-8 incubation time was 1.5 h. (C) Time course of the culture at various cell densities. The cell densities (cells/well) are shown in the graph. Two independent assays were performed in triplicate. The error bars indicate standard deviation.
Please cite this article as: H. Sakamoto, et al., Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.04.003
H. Sakamoto et al. / Parasitology International xxx (2016) xxx–xxx
3
Fig. 3. Drug sensitivities of P. marinus. Tested drugs are indicated beneath each graph. Three independent assays were performed in triplicate. The error bars indicate standard error of the mean.
Fig. 4. Cells transformed with pMOE-mCherry-ble after 2 months of treatment with 100 μg/ml of bleomycin. (A) Cells under fluorescence microscopy. The scale bars indicate 20 μm. (B) Flow cytometry analysis. The gray and magenta histograms indicate wild type and selected transfectants, respectively. (C) Long-term sensitivity to bleomycin. The parasite cells were incubated for 3 weeks in the indicated drug concentrations. The relative growth is calculated as follows: (OD600 of culture with drug − OD600 at day 0)/(OD600 of culture without drug − OD600 at day 0) × 100 [%]. Three independent assays were performed in triplicate. The error bars indicate standard error of the mean.
Please cite this article as: H. Sakamoto, et al., Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.04.003
4
H. Sakamoto et al. / Parasitology International xxx (2016) xxx–xxx
4.2. Drug responses of P. marinus
5. Acknowledgments
Bleomycin strongly inhibits P. marinus growth (IC50 = 1.94 μg/ml); its relative growth in more than 30 μg/ml of bleomycin is almost equal to 0 (Fig. 3). Some reports show that phleomycin is available for selection of T. gondii transfectants [8,9]. Although phleomycin is closely related to bleomycin, its IC50 (38.1 μg/ml) for P. marinus is approximately 20-fold higher than that of bleomycin, and the parasites survive even in a high concentration of phleomycin (Fig. 3). Dihydrofolate reductase inhibitors WR99210 and pyrimethamine are the most often used for Plasmodium spp. (IC50 b 0.05 μM) [10–12] and T. gondii (IC50 b 1 μM) [13]. Additionally, a recent study showed that WR99210 is also available for selecting transfected Babesia bovis [14]. However, WR99210 and pyrimethamine do not inhibit P. marinus cell growth effectively, with IC50 values of 90.3 and 13.3 μM, respectively (Fig. 3).
We thank Dr. José A. Fernández-Robledo, Dr. Shiro Iwanaga and Dr. Takashi Maruyama for supplying the plasmids and Mr. Hideki Sumi and Ms. Toshie Takahashi for providing technical support for MoFlo Astrios EQ. This work was supported by JSPS KAKENHI Grant Number 23117004 and 26253025.
4.3. Bleomycin selection of Sh-ble transfectants Transiently expressed mCherry signals can usually be observed 1 day after transfection. On the other hand, stable transformants will be selected in the presence of bleomycin for several weeks. In the case of the plasmid pMOE-mCherry-ble, mCherry signals were localized in the cytosol of almost all cells (Fig. 4A). Flow cytometry analysis showed that mean fluorescence intensity (MFI) of the treated cells (MFI = 191) was approximately 100-fold higher than that of wildtype cells (MFI = 2.76) (Fig. 4B). The IC50 (N 300 μg/ml) of the line assessed for longterm culture was 2 orders of magnitude higher than that of the parental line (IC50 = 1.07 μg/ml) (Fig. 4C). Note that the mCherry-fused Sh-ble must be expressed in the cytosol; fusion with organelle-localized proteins will cause mislocalization of Sh-ble protein and thus selection failure. 4.4. Conclusion This is the first report of the drug selection system for transfection of P. marinus. We demonstrated that bleomycin enables selection of transfectants. Surprisingly, transfectants were selectively grown in the presence of 100 μg/ml of bleomycin for 2 months even without changing the medium. Transfected cells are extremely easy to obtain with this selection system; manual isolation may be no longer required. We believe that this method facilitates understanding of P. marinus biology and provides new opportunities for functional analyses of proteins in the parasite.
References [1] J.-F. Mangot, D. Debroas, I. Domaizon, Perkinsozoa, a well-known marine protozoan flagellate parasite group, newly identified in lacustrine systems: a review, Hydrobiologia 659 (2010) 37–48. [2] A. Villalba, K.S. Reece, M. Camino Ordás, S.M. Casas, A. Figueras, Perkinsosis in molluscs: a review, Aquat. Living Resour. 17 (2004) 411–432. [3] J.A. Fernández Robledo, E. Caler, M. Matsuzaki, P.J. Keeling, D. Shanmugam, D.S. Roos, et al., The search for the missing link: a relic plastid in Perkinsus? Int. J. Parasitol. 41 (2011) 1217–1229. [4] J.A. Fernández Robledo, Z. Lin, G.R. Vasta, Transfection of the protozoan parasite Perkinsus marinus, Mol. Biochem. Parasitol. 157 (2008) 44–53. [5] T. Kobayashi, N. Morone, T. Kashiyama, H. Oyamada, N. Kurebayashi, T. Murayama, Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research, PLoS One 3 (2008), e3822. [6] C.F. Dungan, R.M. Hamilton, Use of a tetrazolium-based cell proliferation assay to measure effects of in vitro conditions on Perkinsus marinus (Apicomplexa) proliferation, J. Eukaryot. Microbiol. 42 (1995) 379–388. [7] J.D. Gauthier, G.R. Vasta, In Vitro culture of the eastern oyster parasite Perkinsus marinus: optimization of the methodology, J. Invertebr. Pathol. 66 (1995) 156–168. [8] M. Messina, I. Niesman, C. Mercier, L.D. Sibley, Stable DNA transformation of Toxoplasma gondii using phleomycin selection, Gene 165 (1995) 213–217. [9] D. Soldati, K. Kim, J. Kampmeier, J.-F. Dubremetz, J.C. Boothroyd, Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection, Mol. Biochem. Parasitol. 74 (1995) 87–97. [10] D.A. Fidock, T. Nomura, T.E. Wellems, Cycloguanil and its parent compound proguanil demonstrate distinct activities against Plasmodium falciparum malaria parasites transformed with human dihydrofolate reductase, Mol. Pharmacol. 54 (1998) 1140–1147. [11] S. Kinyanjui, E. Mberu, P. Winstanley, D. Jacobus, W. Watkins, The antimalarial triazine WR99210 and the prodrug PS-15: folate reversal of in vitro activity against Plasmodium falciparum and a non-antifolate mode of action of the prodrug, Am.J. Trop. Med. Hyg. 60 (1999) 943–947. [12] L.K. Basco, O. Ramiliarisoa, J. Le Bras, In vitro activity of pyrimethamine, cycloguanil, and other antimalarial drugs against African isolates and clones of Plasmodium falciparum, Am.J.Trop. Med. Hyg. 50 (1994) 193–199. [13] A.J.A.M. van der Ven, E.M.E. Schoondermark-van de Ven, W. Camps, W.J.G. Melchers, P.P. Koopmans, J.W.M. van der Meer, et al., Anti-toxoplasma effect of pyrimethamine, trimethoprim and sulphonamides alone and in combination: implications for therapy, J. Antimicrob. Chemother. 38 (1996) 75–80. [14] M. Asada, M. Tanaka, Y. Goto, N. Yokoyama, N. Inoue, S. Kawazu, Stable expression of green fluorescent protein and targeted disruption of thioredoxin peroxidase-1 gene in Babesia bovis with the WR99210/dhfr selection system, Mol. Biochem. Parasitol. 181 (2012) 162–170.
Please cite this article as: H. Sakamoto, et al., Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.04.003
Contents lists available at ScienceDirect
Parasitology International journal homepage: www.elsevier.com/locate/parint
Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus Hirokazu Sakamoto a,1, Kiyoshi Kita a,b, Motomichi Matsuzaki a,⁎ a b
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, Japan
a r t i c l e
i n f o
Article history: Received 31 December 2015 Received in revised form 15 March 2016 Accepted 5 April 2016 Available online xxxx Keywords: Perkinsus marinus Transfection Drug selection Bleomycin
a b s t r a c t Perkinsus species are notorious unicellular marine parasites that infect commercially important mollusk species including clams and oysters. Recent accumulation of molecular information will greatly facilitate the understanding of Perkinsus biology and development of strategies to control infection. However, the limited availability of methods for genetic manipulation has hindered molecular-based studies of the parasites. In particular, the lack of a drug selection system requires manual isolation of fluorescent cells under a microscope to establish transfected cell lines. Here, we introduce a drug selection system using a glycopeptide antibiotic, bleomycin, and a vector containing the resistance gene Sh-ble. Perkinsus marinus is sensitive to bleomycin, and 100 μg/ml of this drug completely blocks its proliferation. Concomitant expression of Sh-ble enables us to specifically select transfected cells in the presence of the drug. We believe that this system provides new opportunities for functional analyses of this parasite. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Background
2. Objective
Perkinsus is a unicellular marine eukaryote and a member of the Perkinsozoa, a recently recognized group of parasites sister to dinoflagellates [1]. They are notorious for infecting commercially important bivalve species like clams and oysters worldwide [2]. For example, P. marinus causes Dermo disease in the wild and cultivated eastern oyster, Crassostrea viginica, in the eastern coasts of the United States. Perkinsus olseni infects a wide variety of clam species including the Manila clam, Ruditapes philippinarum, and is widespread throughout the tropical and temperate Pacific Ocean as well as the Mediterranean Sea. Methods of controlling diseases caused by these parasites are urgently needed. Beyond the needs of fisheries, Perkinsus spp. are uniquely important in evolutionary biology because they may share a phototroph ancestor with apicomplexan parasites and have independently lost their photosynthetic ability [3]. Comparative analyses between these parasites, especially of the chloroplast remnants, will facilitate understanding of how photosynthetic organisms become parasites. Although a draft genome sequence of P. marinus (DDBJ/ENA/GenBank accession AAXJ01000000) is valuable for this purpose, experimental studies using this molecular information are quite limited, probably because only a small set of genetic manipulation techniques is available [4].
Transgenic techniques are a convincing approach used for functional analysis of proteins of interest. These techniques are currently used in numerous studies of apicomplexan parasites including Plasmodium spp. and Toxoplasma gondii. In P. marinus, however, a drug selection system has not been established, although feasibility of transfection has been reported [4]. If a stably transformed line is required, a small number of cells expressing the transfected gene fused with green fluorescent protein (GFP) must be manually isolated from untransfected cells under a fluorescence microscope. Here, we will introduce a handy assay for parasite growth to screen selection drugs and a selection system using bleomycin (ble) and its resistance gene.
⁎ Corresponding author at: Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. E-mail address: [email protected] (M. Matsuzaki). 1 Present address: Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan.
3. Methods 3.1. Parasites Many strains of Perkinsus spp. are available from American Type Culture Collection (ATCC). We use P. marinus strain CRTW-3HE (ATCC 50439) and maintain it at 26 °C in ATCC medium 1886. Lipid mixture (1000×) (Sigma-Aldrich, Saint Louis, MO) can be used as a substitute for a 100× lipid concentrate indicated in the ATCC's instructions. Instant Ocean Sea Salt (Aquarium Systems, Sarrebourg, France) and Daigo's Artificial Seawater SP (Wako Pure Chemical Industries, Osaka, Japan) can be used as synthetic sea salt. Subculture 1 ml of stationary-phase culture in 10 ml of medium in a T-25 culture flask once per week.
http://dx.doi.org/10.1016/j.parint.2016.04.003 1383-5769/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article as: H. Sakamoto, et al., Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.04.003
2
H. Sakamoto et al. / Parasitology International xxx (2016) xxx–xxx
by site-directed mutagenesis. The coding sequence of PmMOE was replaced with multifunctional GFP (mfGFP) [5] and then mCherry. The bleomycin resistance gene Sh-ble was amplified from the pLEXSY_Ible2 plasmid (Jana Bioscience, Jena, Germany) and inserted as a downstream fusion of mCherry. As a result, the translation product will be mCherry fused with downstream Sh-ble containing an additional Nterminal peptides derived from a multi-cloning site of pColdIII vector (Fig. 1). For transfection, prepare plasmids in more than 1 μg/μl. Fig. 1. A schematic diagram of the transfection plasmid pMOE-mCherry-ble.
3.2. Drug preparation Dissolve bleomycin (Wako Pure Chemical Industries) in water at 10 mg/ml. Filter the solution with an 0.22-μm Ultrafree-MC centrifugal filter unit (Merck Millipore, Billerica, MA) and store the small aliquots at −80 °C until use. 3.3. Drug response assay Dispense 80 μl of culture medium containing 1.0 × 105 log-phase trophozoites per well in a 96-well flat-bottom microtiter plate. Add 20 μl of various concentrations of bleomycin or other drugs to be tested, and incubate the plate at 26 °C for 3 days. Add 10 μl of Cell Counting Kit8 solution (Dojindo Laboratories, Kumamoto, Japan) to each well, and incubate the plate at 26 °C for 1.5 h. Measure the absorbance at 450 nm with a reference wavelength at 600 nm using a microplate reader. We use a SpectraMax M2e-TUY microplate reader (Molecular Devices, LLC, Sunnyvale, CA). Seal the test wells with a sterilized platesealing film to prevent the medium from evaporating during the incubation. On day 0, prepare additional triplicate wells containing the same density of log-phase trophozoites without drug, and measure their absorbance after treatment with Cell Counting Kit-8. Calculate the relative growth as follows: (OD450–600 of culture with drug − OD450–600 at day 0)/(OD450–600 of culture without drug − OD450–600 at day 0) × 100 [%], where OD450–600 means the optical density at 450 nm subtracted with the optical density at 600 nm. Always make triplicates and perform three independent assays. 3.4. Plasmid for transfection The plasmid pMOE-mCherry-ble is available upon request. Construction of this plasmid was performed as follows. We first constructed the transfection plasmid pPmMOE as described in the original report [4]. The two BspHI sites in the backbone were converted to PciI sites
3.5. Transfection and selection of transfectants Harvest 5 × 107 log-phase trophozoites from day 2 or day 3 culture by centrifugation at 800×g for 5 min at 26 °C. Resuspend the cell pellet in 100 μl of Solution 2 supplemented with P2 included in the Basic Parasite Nucleofector Kit 2 (Lonza, Basel, Switzerland). Add 5 μg of supercoiled plasmid, and then perform electroporation using the D023 program in Amaxa Nucleofector II (Lonza) [4]. Dilute the cells with 2 ml of culture medium, dispense them into two wells in a 6well plate, and incubate at 26 °C for several days. Confirm transiently expressed mCherry signal, and then dilute 50 μl of the culture in 1 ml of the medium containing 100 μg/ml of bleomycin in another well. Tightly seal the plate with a sterilized plate-sealing film and incubate for up to two months without changing the medium. 3.6. Flow cytometry analysis (optional) Dilute P. marinus cells to less than 1 × 106 cells/ml in 1 ml of ATCC medium 1886 without lipid mixture, JLP carbohydrate, and fetal bovine serum. Analyze the cells with a flow cytometer after passing cell suspension through a 35-μm nylon mesh cell strainer (Becton Dickinson, Franklin Lakes, NJ). 4. Special remarks/comments 4.1. The drug response assay system This assay system is similar to the method previously reported for P. marinus [6], although the tetrazolium salt used is different. Cell Counting Kit-8 requires optimization of the incubation period for cell number determination. The 1.5-h incubation gives us the best correlation between absorbance and cell number (Fig. 2A), but it may depend on species, strain, culture medium, and temperature. The linearity range was 1 × 106 to 1 × 107 cells/well (R2 = 0.9995) in our experimental setup (Fig. 2B). Seeding density is critical (Fig. 2C) because the growth rate of P. marinus depends on an autocrine factor and therefore cell density [7]. We used a seeding density of 1 × 105 cells/well and a culture time of 3 days.
Fig. 2. Effect of parameters on the cell viability assay. (A) Effect of incubation time on the correlation between cell numbers and the optical density (OD) values. The incubation times (h) are shown in the graph. (B) Regression between the OD value and cell number within the 1 × 106 to 1 × 107 cells/well range when the WST-8 incubation time was 1.5 h. (C) Time course of the culture at various cell densities. The cell densities (cells/well) are shown in the graph. Two independent assays were performed in triplicate. The error bars indicate standard deviation.
Please cite this article as: H. Sakamoto, et al., Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.04.003
H. Sakamoto et al. / Parasitology International xxx (2016) xxx–xxx
3
Fig. 3. Drug sensitivities of P. marinus. Tested drugs are indicated beneath each graph. Three independent assays were performed in triplicate. The error bars indicate standard error of the mean.
Fig. 4. Cells transformed with pMOE-mCherry-ble after 2 months of treatment with 100 μg/ml of bleomycin. (A) Cells under fluorescence microscopy. The scale bars indicate 20 μm. (B) Flow cytometry analysis. The gray and magenta histograms indicate wild type and selected transfectants, respectively. (C) Long-term sensitivity to bleomycin. The parasite cells were incubated for 3 weeks in the indicated drug concentrations. The relative growth is calculated as follows: (OD600 of culture with drug − OD600 at day 0)/(OD600 of culture without drug − OD600 at day 0) × 100 [%]. Three independent assays were performed in triplicate. The error bars indicate standard error of the mean.
Please cite this article as: H. Sakamoto, et al., Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.04.003
4
H. Sakamoto et al. / Parasitology International xxx (2016) xxx–xxx
4.2. Drug responses of P. marinus
5. Acknowledgments
Bleomycin strongly inhibits P. marinus growth (IC50 = 1.94 μg/ml); its relative growth in more than 30 μg/ml of bleomycin is almost equal to 0 (Fig. 3). Some reports show that phleomycin is available for selection of T. gondii transfectants [8,9]. Although phleomycin is closely related to bleomycin, its IC50 (38.1 μg/ml) for P. marinus is approximately 20-fold higher than that of bleomycin, and the parasites survive even in a high concentration of phleomycin (Fig. 3). Dihydrofolate reductase inhibitors WR99210 and pyrimethamine are the most often used for Plasmodium spp. (IC50 b 0.05 μM) [10–12] and T. gondii (IC50 b 1 μM) [13]. Additionally, a recent study showed that WR99210 is also available for selecting transfected Babesia bovis [14]. However, WR99210 and pyrimethamine do not inhibit P. marinus cell growth effectively, with IC50 values of 90.3 and 13.3 μM, respectively (Fig. 3).
We thank Dr. José A. Fernández-Robledo, Dr. Shiro Iwanaga and Dr. Takashi Maruyama for supplying the plasmids and Mr. Hideki Sumi and Ms. Toshie Takahashi for providing technical support for MoFlo Astrios EQ. This work was supported by JSPS KAKENHI Grant Number 23117004 and 26253025.
4.3. Bleomycin selection of Sh-ble transfectants Transiently expressed mCherry signals can usually be observed 1 day after transfection. On the other hand, stable transformants will be selected in the presence of bleomycin for several weeks. In the case of the plasmid pMOE-mCherry-ble, mCherry signals were localized in the cytosol of almost all cells (Fig. 4A). Flow cytometry analysis showed that mean fluorescence intensity (MFI) of the treated cells (MFI = 191) was approximately 100-fold higher than that of wildtype cells (MFI = 2.76) (Fig. 4B). The IC50 (N 300 μg/ml) of the line assessed for longterm culture was 2 orders of magnitude higher than that of the parental line (IC50 = 1.07 μg/ml) (Fig. 4C). Note that the mCherry-fused Sh-ble must be expressed in the cytosol; fusion with organelle-localized proteins will cause mislocalization of Sh-ble protein and thus selection failure. 4.4. Conclusion This is the first report of the drug selection system for transfection of P. marinus. We demonstrated that bleomycin enables selection of transfectants. Surprisingly, transfectants were selectively grown in the presence of 100 μg/ml of bleomycin for 2 months even without changing the medium. Transfected cells are extremely easy to obtain with this selection system; manual isolation may be no longer required. We believe that this method facilitates understanding of P. marinus biology and provides new opportunities for functional analyses of proteins in the parasite.
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Please cite this article as: H. Sakamoto, et al., Drug selection using bleomycin for transfection of the oyster-infecting parasite Perkinsus marinus, Parasitology International (2016), http://dx.doi.org/10.1016/j.parint.2016.04.003