- Email: [email protected]
Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei
Accepted Manuscript Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei Sergey A. Timofeev, Igor V. Senderskiy, Alexander A. Tsarev, Yuri S. Tokarev, Viacheslav V. Dolgikh PII: DOI: Reference:
S0022-2011(16)30242-7 http://dx.doi.org/10.1016/j.jip.2016.12.002 YJIPA 6896
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
6 June 2016 11 November 2016 11 December 2016
Please cite this article as: Timofeev, S.A., Senderskiy, I.V., Tsarev, A.A., Tokarev, Y.S., Dolgikh, V.V., Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei, Journal of Invertebrate Pathology (2016), doi: http://dx.doi.org/10.1016/j.jip.2016.12.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei.
Authors and Affiliations Sergey A.Timofeev, Igor V. Senderskiy, Alexander A. Tsarev, Yuri S. Tokarev, Viacheslav V. Dolgikh*.
Laboratory of Microbiological Control, All-Russian Institute for Plant Protection, St. Petersburg, Pushkin, Russia.
* Corresponding author. Mailing address: Laboratory of Microbiological Control, All-Russian Institute for Plant Protection, Podbelskogo shosse, 3, 196608, St. Petersburg-Pushkin, Russia. Phone: +7 812 470 43 84. Fax: +7 812 470 51 10. E-mail: [email protected].
1
Abstract Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study.
Keywords: insect pathogens, intracellular parasites, microsporidia, Paranosema locustae, secretory proteins, hexokinase, heterologous expression, nuclear localization.
1. Introduction
Microsporidia are a large group of intracellular parasites related to fungi that infect a wide range of animals and some protozoan species (Vivier, 1975; Scheid, 2007). Widely distributed in insects, microsporidia are considered to be long-term regulators of insect populations and may suppress outbreaks of herbivore pests (Bjornson, Oi, 2014). Paranosema (Nosema, Antonospora) locustae, an orthopteran pathogen, is the only microsporidian species produced as commercial product for biological control (Henry et al., 1985). The chronic nature of microsporidian infections and their metabolic dependence on infected host cells suggest that these parasites should finely regulate host metabolic pathways to ensure a sufficient supply of nutrients for replication and sporogenesis (Williams et al., 2014). In addition, intracellular pathogens must resist immune responses of host cell. Molecular mechanisms of the effects on the host produced 2
by microsporidian infection remain unidentified. Like intracellular apicomplexans (Gilbert et al., 2007; Schmuckli-Maurer et al., 2009) and kinetoplastids (Nandan, Reiner, 2005; Costa et al., 2016), microsporidia probably secrete a variety of proteins affecting expression of host genes or interacting with host cell signaling pathways. Hexokinase (Hxk) is considered to be one of the most interesting proteins secreted into infected cells by microsporidia as there is good reason to believe that it is involved in the regulation of transcriptional activity of genes in nuclei of infected host cells. Hxk2 is a bifunctional protein in the yeast Saccharomyces cerevisiae and in cancer Hela cells, demonstrating dual nucleocytoplasmic cellular localization (Neary, Pastorino, 2010). In yeast nuclei, Hxk2 has been implicated in processes of glucose repression and induction of several genes of carbohydrate metabolism (Petit et al., 2000; Moreno, Herrero, 2002). Like the yeast enzyme, microsporidian Hxk may control the expression of host carbohydrate metabolism genes in infected host cells. Unlike other glycolytic enzymes, Hxk of the microsporidian Nematocida parisii is highly expressed at early stages of parasite intracellular development in Caenorhabdus elegans (Cuomo et al., 2012). Additionally, Hxks signal peptides of six microsporidian species were shown to cause secretion of reporter enzyme in the S. cerevisiae secretion trap system (Cuomo et al., 2012). Previously, we demonstrated secretion of P. locustae Hxk into infected adipocytes of Locusta migratoria and accumulation in the host cell nuclei (Senderskiy et al., 2014). In this study, P. locustae Hxk without N-terminal 12 aa peptide (to prevent entry into the secretory pathway) was expressed in the yeast Pichia pastoris and in lepidopteran (Spodoptera frugiperda) Sf9 cells. During the course of expression, heterologous protein accumulated in the nuclei of insect cells but not in yeast cell nuclei.
2. Materials and methods
3
2.1. DNA constructs for expression of P. locustae protein.
The sequence encoding P. locustae Hxk without N-terminal 12 aa peptide was amplified by PCR using Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific) and previously cloned gene copies (Senderskiy et al., 2014). To insert DNA fragments into vector pPIC3.5 (Thermo Fisher Scientific), the primers ccaaAGATCTATGTCATTCAACATGCAGTGCGATGA (forward) and gtcaGAATTCTACTCAACTAAGAAGGAAGC (reverse) containing BglII/ EcoRI sites (underlined) were used. To insert the Hxk gene into a pIEx-4 plasmid (Merck Millipore), the primers ccaaCCATGGGTTCATTCAACATGCAGTGCGATGA (forward) and gtcaCTGCAGCTACTCAACTAAGAAGGAAGCGTAT (reverse) containing NcoI/PstI sites (underlined) were used. The PCR products were gel purified, cleaved with the restriction enzymes and inserted into vector pPIC3.5 digested with BamHI/EcoRI or into vector pIEx-4 linearized with NcoI/PstI.
2.2. Heterologous expression and immunolocalization of A. locustae Hxk in P. pastoris.
P. locustae Hxk was expressed in P. pastoris (strain GS115) as previously described (Senderskiy et al., 2014). To inhibit protein export from the nuclei, Leptomycin B (SigmaAldrich) was added 4 hours before the end of expression to a final concentration 50 nM. After expression, P. pastoris cells were fixed for 1 h with 4 % paraformaldehyde in PBS (138 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, pH 6.8, pH 7.4), washed and spheroplasted in PBS containing 1.2 M sorbitol, 1.5% β-mercaptoethanol and 100 U/ml zymolyase (Thermo Fisher Scientific) overnight at 30°C. Cells were washed with PBS, transferred onto slides coated with polylysine and incubated in TTBS (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 4
7.4) with 1% bovine serum albumin (BSA) for 1 h at room temperature. The cells were then incubated overnight at 4 ºC with diluted 1:50 in TTBS polyclonal antibodies (Abs), which were previously raised and purified against recombinant P. locustae Hxk (Senderskiy et al., 2014). After washing with TTBS, slides were incubated for 2 h at room temperature with Alexa Fluor 488 - conjugated Anti-Rabbit Abs (Thermo Fisher Scientific) diluted 1:50 in TTBS and washed with the same solution. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). The preparations in Vectashield mounting medium (Vector Laboratories) were analyzed with Leica TCS SP5 MP confocal microscope.
2.3. Heterologous expression and immunolocalization of P. locustae Hxk in Sf9 cells.
SF9 cells were maintained in SF900 III medium (Thermo Fisher Scientific) at 27C and transfected by plasmid DNA using Insect Gene Juice Transfection Reagent (Merck Millipore) according to the manufacturer’s protocol. After cultivation for 48 h cells were fixed for 2 h in PBS with 4% paraformaldehyde, washed with PBS, blocked and permeabilized in TTBS with 1% BSA for 1 h at room temperature. Immunolabeling was performed as described above for P. pastoris. The preparations in Vectashield mounting medium were analyzed with Carl Zeiss AxioImager M1 fluorescent microscope. The following controls were used: cells not subjected to transfection, transfected cells not treated with Abs, transfected cells treated only with first Abs against P. locustae Hxk or only with second Alexa Fluor 488 - conjugated anti-Rabbit Abs.
3. Results
Heterologous expression of P. locustae Hxk in methylotrophic yeast P. pastoris was not accompanied by nuclear localization of parasite protein (Fig. 1). The addition of leptomycin B, a 5
specific inhibitor of nuclear export in fission yeast Schizosaccharomyces pombe (Nishi et al., 1994) and mammalian cells (Kudo et al., 1998), into cultivation medium also did not result in accumulation of parasite Hxk in the nuclei. Immunolabeling of Sf9 cells expressing P. locustae Hxk showed specific accumulation of the heterologous protein in insect nuclei even without Leptomycin B blocking of nuclear export (Fig. 2, a-d). The clear co-localization of hexokinase with the nuclei of Sf9 cells was confirmed by DAPI staining. No specific labeling of nuclei was observed in the control preparations (Fig. 2, d-e).
4. Discussion In this study, microsporidian Hxk without N-terminal 12 aа signal peptide was expressed in yeasts and in insect cells in order to simulate the process of its secretion from microsporidia into the host cytoplasm. Despite the conservatism of the nuclear import apparatus (Macara, 2001), yeast cells did not recognize P. locustae Hxk as a nuclear protein. In contrast, the enzyme clearly accumulated in the nuclei of insect Sf9 cells. This result is in agreement with our previous data illustrating nuclear localization of P. locustae Hxk in infected host cells (Senderskiy et al., 2014) and demonstrates that cell lines derived from animals related to the natural hosts are the most suitable models to investigate molecular interactions between intracellular pathogens and their hosts. The physiological role of microsporidian Hxk in host nuclei may be associated with upregulation of glucose (hexoses) uptake by infected cells. It would intensify the replication and sporulation of parasites by more ATP production in host mitochondria (Hacker et al., 2014). Besides, during sporogenesis microsporidia need a lot of carbohydrates for trehalose storing as well as for formation of chitin-rich spore wall and O-mannosylated polar tube (Xu et al., 2004). There is some evidence that yeast Hxk2 regulates transcriptional activity of genes encoding 6
hexose carriers diminishing expression of high-affinity and elevating expression of low-affinity transporters in the presence of high glucose concentration (Petit et al., 2000). Further analysis of expression of hexose transporters or glucose uptake in infected by microsporidia or Hxkexpressing cells should verify this hypothesis.
Acknowledgments We acknowledge financial support from the Russian Foundation of Basic Research (RFBR 1504-04968, 15-34-20567).
7
References Bjornson, S., Oi, D., 2014. Microsporidia biological control agents and pathogens of beneficial insects, in: Weiss, L.M., Becnel, J.J. (Eds.), Microsporidia: pathogens of opportunity. Wiley-Blackwell, pp. 635-670. Costa, R.W., da Silveira, J.F., Bahia, D. 2016. Interactions between Trypanosoma cruzi secreted proteins and host cell signaling pathways. Front Microbiol. 7, 388. Cuomo, C.A. Desjardins, C.A., Bakowski, M.A., Goldberg, J., Ma, A.T., Becnel, J.J., Didier, E.S., Fan, L., Heiman, D.I., Levin, J.Z., Young, S., Zeng, Q., Troemel E.R., 2012. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome. Res. 22, 2478–2488. Hacker, C., Howell, M., Bhella, D., Lucocq, J. 2014. Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC. Cell Microbiol. 16, 565-579. Henry, J.E., 1985. Effect of grasshopper species, cage density, light-intensity, and method of inoculation on mass-production of Nosema locustae (Microsporida: Nosematidae). J. Econ. Entomol. 78, 1245–1250. Gilbert, L.A., Ravindran, S., Turetzky, J.M., Boothroyd, J.C., Bradley, P.J., 2007. Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells. Eukaryot. Cell. 6, 73–83. Kudo, N., Wolff, B., Sekimoto, T., Schreiner, E.P., Yoneda, Y., Yanagida, M., Horinouchi, S., Yoshida, M., 1998. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242, 540–547. Macara, I.G., 2010. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570– 594. 8
Moreno, F., Herrero, P., 2002. The hexokinase 2-dependent glucose signal transduction pathway of Saccharomyces cerevisiae. FEMS Microbiol. Rev. 26, 83–90. Nandan, D., Reiner, N.E., 2005. Leishmania donovani engages in regulatory interference by targeting macrophage protein tyrosine phosphatase SHP-1. Clin. Immunol. 114, 266–277. Neary, C.L., Pastorino J.G., 2010. Nucleocytoplasmic shuttling of hexokinase II in a cancer cell. Biochem. Biophys. Res. Commun. 394, 1075–1081. Nishi, K., Yoshida, M., Fujiwara, D., Nishikawa, M., Horinouchi, S., Beppu, T., 1994. Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J. Biol. Chem. 269, 6320–6324. Petit, T., Diderich, J.A., Kruckeberg, A.L., Gancedo, C., Van Dam, K., 2000. Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae. J. Bacteriol. 182, 6815–6818. Scheid, P., 2007. Mechanism of intrusion of a microspordian-like organism into the nucleus of host amoebae (Vannella sp.) isolated from a keratitis patient. Parasitol. Res. 101, 1097– 1102. Schmuckli-Maurer, J., Casanova, C., Schmied, S., Affentranger, S., Parvanova, I., Kang'a, S., Nene, V., Katzer, F., McKeever, D., Müller, J., Bishop, R., Pain, A., Dobbelaere, D.A.E., 2009.) Expression analysis of the Theileria parva subtelomere-encoded variable secreted protein gene family. PLoS One 4, e4839. Senderskiy, I.V., Timofeev, S.A., Seliverstova, E.V., Pavlova, O.A., Dolgikh, V.V., 2014. Secretion of Antonospora (Paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes. PLoS One 9, e93585. Vivier, E., 1975. The microsporidia of the protozoa. Protistology 11, 345–361. 9
Williams, B.A.P., Dolgikh, V.V., Sokolova, Y.Y., 2014. Microsporidian biochemistry and physiology, Weiss, L.M., Becnel, J.J. (Eds.), Microsporidia: pathogens of opportunity. Wiley-Blackwell, pp. 245-260. Xu, Y., Takvorian P.M., Cali A., Orr G., Weiss L.M., 2004. Glycosylation of the major polar tube protein of Encephalitozoon hellem, a microsporidian parasite that infects humans. Infect Immun. 72, 6341-6350.
10
Figure legends Fig. 1. Immunolocalization of Paranosema locustae Hxk expressed in Pichia pastoris. Abs against parasite protein showed Hxk accumulation outside the host cell nuclei stained with DAPI. White arrows on the left images point to dark nuclei not stained with anti-Hxk Abs. Scale bars, 5 µm.
Fig. 2. Immunolocalization of P. locustae Hxk expressed in Spodoptera frugiperda Sf9 cells. Staining of cells with anti-Hxk Abs and DAPI demonstrated clear localization of the microsporidian protein within the insect cell nuclei. a-c. Single cells transformed with plasmid expressing P. locustae Hxk. d. Hxk-expressing and non-expressing cells in the same culture. e. Control (non-transformed) Sf9 cells stained with anti-Hxk Abs. Scale bars, 10 µm.
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13
Highlights
Hexokinase secreted by microsporidia into host cells is accumulated in the nuclei.
Paranosema locustae hexokinase shows similar behavior in the host and Sf9 cells.
Sf9 cell line is a suitable model to study proteins secreted by insect microsporidia.
14
S0022-2011(16)30242-7 http://dx.doi.org/10.1016/j.jip.2016.12.002 YJIPA 6896
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
6 June 2016 11 November 2016 11 December 2016
Please cite this article as: Timofeev, S.A., Senderskiy, I.V., Tsarev, A.A., Tokarev, Y.S., Dolgikh, V.V., Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei, Journal of Invertebrate Pathology (2016), doi: http://dx.doi.org/10.1016/j.jip.2016.12.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei.
Authors and Affiliations Sergey A.Timofeev, Igor V. Senderskiy, Alexander A. Tsarev, Yuri S. Tokarev, Viacheslav V. Dolgikh*.
Laboratory of Microbiological Control, All-Russian Institute for Plant Protection, St. Petersburg, Pushkin, Russia.
* Corresponding author. Mailing address: Laboratory of Microbiological Control, All-Russian Institute for Plant Protection, Podbelskogo shosse, 3, 196608, St. Petersburg-Pushkin, Russia. Phone: +7 812 470 43 84. Fax: +7 812 470 51 10. E-mail: [email protected].
1
Abstract Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study.
Keywords: insect pathogens, intracellular parasites, microsporidia, Paranosema locustae, secretory proteins, hexokinase, heterologous expression, nuclear localization.
1. Introduction
Microsporidia are a large group of intracellular parasites related to fungi that infect a wide range of animals and some protozoan species (Vivier, 1975; Scheid, 2007). Widely distributed in insects, microsporidia are considered to be long-term regulators of insect populations and may suppress outbreaks of herbivore pests (Bjornson, Oi, 2014). Paranosema (Nosema, Antonospora) locustae, an orthopteran pathogen, is the only microsporidian species produced as commercial product for biological control (Henry et al., 1985). The chronic nature of microsporidian infections and their metabolic dependence on infected host cells suggest that these parasites should finely regulate host metabolic pathways to ensure a sufficient supply of nutrients for replication and sporogenesis (Williams et al., 2014). In addition, intracellular pathogens must resist immune responses of host cell. Molecular mechanisms of the effects on the host produced 2
by microsporidian infection remain unidentified. Like intracellular apicomplexans (Gilbert et al., 2007; Schmuckli-Maurer et al., 2009) and kinetoplastids (Nandan, Reiner, 2005; Costa et al., 2016), microsporidia probably secrete a variety of proteins affecting expression of host genes or interacting with host cell signaling pathways. Hexokinase (Hxk) is considered to be one of the most interesting proteins secreted into infected cells by microsporidia as there is good reason to believe that it is involved in the regulation of transcriptional activity of genes in nuclei of infected host cells. Hxk2 is a bifunctional protein in the yeast Saccharomyces cerevisiae and in cancer Hela cells, demonstrating dual nucleocytoplasmic cellular localization (Neary, Pastorino, 2010). In yeast nuclei, Hxk2 has been implicated in processes of glucose repression and induction of several genes of carbohydrate metabolism (Petit et al., 2000; Moreno, Herrero, 2002). Like the yeast enzyme, microsporidian Hxk may control the expression of host carbohydrate metabolism genes in infected host cells. Unlike other glycolytic enzymes, Hxk of the microsporidian Nematocida parisii is highly expressed at early stages of parasite intracellular development in Caenorhabdus elegans (Cuomo et al., 2012). Additionally, Hxks signal peptides of six microsporidian species were shown to cause secretion of reporter enzyme in the S. cerevisiae secretion trap system (Cuomo et al., 2012). Previously, we demonstrated secretion of P. locustae Hxk into infected adipocytes of Locusta migratoria and accumulation in the host cell nuclei (Senderskiy et al., 2014). In this study, P. locustae Hxk without N-terminal 12 aa peptide (to prevent entry into the secretory pathway) was expressed in the yeast Pichia pastoris and in lepidopteran (Spodoptera frugiperda) Sf9 cells. During the course of expression, heterologous protein accumulated in the nuclei of insect cells but not in yeast cell nuclei.
2. Materials and methods
3
2.1. DNA constructs for expression of P. locustae protein.
The sequence encoding P. locustae Hxk without N-terminal 12 aa peptide was amplified by PCR using Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific) and previously cloned gene copies (Senderskiy et al., 2014). To insert DNA fragments into vector pPIC3.5 (Thermo Fisher Scientific), the primers ccaaAGATCTATGTCATTCAACATGCAGTGCGATGA (forward) and gtcaGAATTCTACTCAACTAAGAAGGAAGC (reverse) containing BglII/ EcoRI sites (underlined) were used. To insert the Hxk gene into a pIEx-4 plasmid (Merck Millipore), the primers ccaaCCATGGGTTCATTCAACATGCAGTGCGATGA (forward) and gtcaCTGCAGCTACTCAACTAAGAAGGAAGCGTAT (reverse) containing NcoI/PstI sites (underlined) were used. The PCR products were gel purified, cleaved with the restriction enzymes and inserted into vector pPIC3.5 digested with BamHI/EcoRI or into vector pIEx-4 linearized with NcoI/PstI.
2.2. Heterologous expression and immunolocalization of A. locustae Hxk in P. pastoris.
P. locustae Hxk was expressed in P. pastoris (strain GS115) as previously described (Senderskiy et al., 2014). To inhibit protein export from the nuclei, Leptomycin B (SigmaAldrich) was added 4 hours before the end of expression to a final concentration 50 nM. After expression, P. pastoris cells were fixed for 1 h with 4 % paraformaldehyde in PBS (138 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, pH 6.8, pH 7.4), washed and spheroplasted in PBS containing 1.2 M sorbitol, 1.5% β-mercaptoethanol and 100 U/ml zymolyase (Thermo Fisher Scientific) overnight at 30°C. Cells were washed with PBS, transferred onto slides coated with polylysine and incubated in TTBS (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 4
7.4) with 1% bovine serum albumin (BSA) for 1 h at room temperature. The cells were then incubated overnight at 4 ºC with diluted 1:50 in TTBS polyclonal antibodies (Abs), which were previously raised and purified against recombinant P. locustae Hxk (Senderskiy et al., 2014). After washing with TTBS, slides were incubated for 2 h at room temperature with Alexa Fluor 488 - conjugated Anti-Rabbit Abs (Thermo Fisher Scientific) diluted 1:50 in TTBS and washed with the same solution. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). The preparations in Vectashield mounting medium (Vector Laboratories) were analyzed with Leica TCS SP5 MP confocal microscope.
2.3. Heterologous expression and immunolocalization of P. locustae Hxk in Sf9 cells.
SF9 cells were maintained in SF900 III medium (Thermo Fisher Scientific) at 27C and transfected by plasmid DNA using Insect Gene Juice Transfection Reagent (Merck Millipore) according to the manufacturer’s protocol. After cultivation for 48 h cells were fixed for 2 h in PBS with 4% paraformaldehyde, washed with PBS, blocked and permeabilized in TTBS with 1% BSA for 1 h at room temperature. Immunolabeling was performed as described above for P. pastoris. The preparations in Vectashield mounting medium were analyzed with Carl Zeiss AxioImager M1 fluorescent microscope. The following controls were used: cells not subjected to transfection, transfected cells not treated with Abs, transfected cells treated only with first Abs against P. locustae Hxk or only with second Alexa Fluor 488 - conjugated anti-Rabbit Abs.
3. Results
Heterologous expression of P. locustae Hxk in methylotrophic yeast P. pastoris was not accompanied by nuclear localization of parasite protein (Fig. 1). The addition of leptomycin B, a 5
specific inhibitor of nuclear export in fission yeast Schizosaccharomyces pombe (Nishi et al., 1994) and mammalian cells (Kudo et al., 1998), into cultivation medium also did not result in accumulation of parasite Hxk in the nuclei. Immunolabeling of Sf9 cells expressing P. locustae Hxk showed specific accumulation of the heterologous protein in insect nuclei even without Leptomycin B blocking of nuclear export (Fig. 2, a-d). The clear co-localization of hexokinase with the nuclei of Sf9 cells was confirmed by DAPI staining. No specific labeling of nuclei was observed in the control preparations (Fig. 2, d-e).
4. Discussion In this study, microsporidian Hxk without N-terminal 12 aа signal peptide was expressed in yeasts and in insect cells in order to simulate the process of its secretion from microsporidia into the host cytoplasm. Despite the conservatism of the nuclear import apparatus (Macara, 2001), yeast cells did not recognize P. locustae Hxk as a nuclear protein. In contrast, the enzyme clearly accumulated in the nuclei of insect Sf9 cells. This result is in agreement with our previous data illustrating nuclear localization of P. locustae Hxk in infected host cells (Senderskiy et al., 2014) and demonstrates that cell lines derived from animals related to the natural hosts are the most suitable models to investigate molecular interactions between intracellular pathogens and their hosts. The physiological role of microsporidian Hxk in host nuclei may be associated with upregulation of glucose (hexoses) uptake by infected cells. It would intensify the replication and sporulation of parasites by more ATP production in host mitochondria (Hacker et al., 2014). Besides, during sporogenesis microsporidia need a lot of carbohydrates for trehalose storing as well as for formation of chitin-rich spore wall and O-mannosylated polar tube (Xu et al., 2004). There is some evidence that yeast Hxk2 regulates transcriptional activity of genes encoding 6
hexose carriers diminishing expression of high-affinity and elevating expression of low-affinity transporters in the presence of high glucose concentration (Petit et al., 2000). Further analysis of expression of hexose transporters or glucose uptake in infected by microsporidia or Hxkexpressing cells should verify this hypothesis.
Acknowledgments We acknowledge financial support from the Russian Foundation of Basic Research (RFBR 1504-04968, 15-34-20567).
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References Bjornson, S., Oi, D., 2014. Microsporidia biological control agents and pathogens of beneficial insects, in: Weiss, L.M., Becnel, J.J. (Eds.), Microsporidia: pathogens of opportunity. Wiley-Blackwell, pp. 635-670. Costa, R.W., da Silveira, J.F., Bahia, D. 2016. Interactions between Trypanosoma cruzi secreted proteins and host cell signaling pathways. Front Microbiol. 7, 388. Cuomo, C.A. Desjardins, C.A., Bakowski, M.A., Goldberg, J., Ma, A.T., Becnel, J.J., Didier, E.S., Fan, L., Heiman, D.I., Levin, J.Z., Young, S., Zeng, Q., Troemel E.R., 2012. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome. Res. 22, 2478–2488. Hacker, C., Howell, M., Bhella, D., Lucocq, J. 2014. Strategies for maximizing ATP supply in the microsporidian Encephalitozoon cuniculi: direct binding of mitochondria to the parasitophorous vacuole and clustering of the mitochondrial porin VDAC. Cell Microbiol. 16, 565-579. Henry, J.E., 1985. Effect of grasshopper species, cage density, light-intensity, and method of inoculation on mass-production of Nosema locustae (Microsporida: Nosematidae). J. Econ. Entomol. 78, 1245–1250. Gilbert, L.A., Ravindran, S., Turetzky, J.M., Boothroyd, J.C., Bradley, P.J., 2007. Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected host cells. Eukaryot. Cell. 6, 73–83. Kudo, N., Wolff, B., Sekimoto, T., Schreiner, E.P., Yoneda, Y., Yanagida, M., Horinouchi, S., Yoshida, M., 1998. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell Res. 242, 540–547. Macara, I.G., 2010. Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570– 594. 8
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Figure legends Fig. 1. Immunolocalization of Paranosema locustae Hxk expressed in Pichia pastoris. Abs against parasite protein showed Hxk accumulation outside the host cell nuclei stained with DAPI. White arrows on the left images point to dark nuclei not stained with anti-Hxk Abs. Scale bars, 5 µm.
Fig. 2. Immunolocalization of P. locustae Hxk expressed in Spodoptera frugiperda Sf9 cells. Staining of cells with anti-Hxk Abs and DAPI demonstrated clear localization of the microsporidian protein within the insect cell nuclei. a-c. Single cells transformed with plasmid expressing P. locustae Hxk. d. Hxk-expressing and non-expressing cells in the same culture. e. Control (non-transformed) Sf9 cells stained with anti-Hxk Abs. Scale bars, 10 µm.
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Highlights
Hexokinase secreted by microsporidia into host cells is accumulated in the nuclei.
Paranosema locustae hexokinase shows similar behavior in the host and Sf9 cells.
Sf9 cell line is a suitable model to study proteins secreted by insect microsporidia.
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