Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
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A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1

  • Xiao, Bin (State Key Laboratory of Organ Failure, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Biotechnology, Southern Medical University) ;
  • Kuang, Zhenzhan (State Key Laboratory of Organ Failure, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Biotechnology, Southern Medical University) ;
  • Zhan, Yanli (State Key Laboratory of Organ Failure, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Biotechnology, Southern Medical University) ;
  • Chen, Daxiang (State Key Laboratory of Organ Failure, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Biotechnology, Southern Medical University) ;
  • Gao, Yang (State Key Laboratory of Organ Failure, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Biotechnology, Southern Medical University) ;
  • Li, Ming (State Key Laboratory of Organ Failure, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Biotechnology, Southern Medical University) ;
  • Luo, Shuhong (State Key Laboratory of Organ Failure, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Biotechnology, Southern Medical University) ;
  • Hao, Wenbo (State Key Laboratory of Organ Failure, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Biotechnology, Southern Medical University)
  • Received : 2015.11.03
  • Accepted : 2015.12.29
  • Published : 2016.02.29
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Abstract

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of $Na^+$ and $H^+$ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of $Ca^{2+}$. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.

Keywords

References

  1. Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004; 363: 1965-1976. https://doi.org/10.1016/S0140-6736(04)16412-X
  2. Kaye A. Toxoplasmosis: diagnosis, treatment, and prevention in congenitally exposed infants. J Pediatr Health Care 2011; 25: 355-364. https://doi.org/10.1016/j.pedhc.2010.04.008
  3. Arrizabalaga G, Boothroyd JC. Role of calcium during Toxoplasma gondii invasion and egress. Int J Parasitol 2004; 34: 361-368. https://doi.org/10.1016/j.ijpara.2003.11.017
  4. Karasov AO, Boothroyd JC, Arrizabalaga G. Identification and disruption of a rhoptry-localized homologue of sodium hydrogen exchangers in Toxoplasma gondii. Int J Parasitol 2005; 35: 285-291. https://doi.org/10.1016/j.ijpara.2004.11.015
  5. Francia ME, Wicher S, Pace DA, Sullivan J, Moreno SN, Arrizabalaga G. A Toxoplasma gondii protein with homology to intracellular type $Na^+$/$H^+$ exchangers is important for osmoregulation and invasion. Exp Cell Res 2011; 317: 1382-1396. https://doi.org/10.1016/j.yexcr.2011.03.020
  6. Roiko MS, Svezhova N, Carruthers VB. Acidification activates Toxoplasma gondii motility and egress by enhancing protein secretion and cytolytic activity. PLoS Pathog 2014; 10: e1004488. https://doi.org/10.1371/journal.ppat.1004488
  7. Carruthers VB, Sibley LD. Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol Microbiol 1999; 31: 421-428. https://doi.org/10.1046/j.1365-2958.1999.01174.x
  8. Carruthers VB, Moreno SN, Sibley LD. Ethanol and acetaldehyde elevate intracellular [$Ca^{2+}$] and stimulate microneme discharge in Toxoplasma gondii. Biochem J 1999; 342 (Pt 2): 379-386. https://doi.org/10.1042/bj3420379
  9. Lin J, Lin X, Yang GH, Wang Y, Peng BW, Lin JY. Toxoplasma gondii: expression of GRA1 gene in endoplasmic reticulum promotes both growth and adherence and modulates intracellular calcium release in macrophages. Exp Parasitol 2010; 125: 165-171. https://doi.org/10.1016/j.exppara.2010.01.010
  10. Plattner H, Sehring IM, Mohamed IK, Kiranda K, De Souza W, Billington R, Genazzani A, Ladenburger EM. Calcium signaling in closely related protozoan groups (Alveolata): non-parasitic ciliates (Paramecium, Tetrahymena) vs. parasitic Apicomplexa (Plasmodium, Toxoplasma). Cell Calcium 2012; 51: 351-382. https://doi.org/10.1016/j.ceca.2012.01.006
  11. Wu L, Chen SX, Jiang XG, Fu XL, Shen YJ,Cao JP. Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems. Exp Parasitol 2012; 130: 91-94. https://doi.org/10.1016/j.exppara.2011.10.006
  12. Barbosa BF, Gomes AO, Ferro EA, Napolitano DR, Mineo JR, Silva NM. Enrofloxacin is able to control Toxoplasma gondii infection in both in vitro and in vivo experimental models. Vet Parasitol 2012; 187: 44-52. https://doi.org/10.1016/j.vetpar.2011.12.039
  13. Pappas G, Roussos N, Falagas ME. Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. Int J Parasitol 2009; 39: 1385-1394. https://doi.org/10.1016/j.ijpara.2009.04.003
  14. Arrizabalaga G, Ruiz F, Moreno S, Boothroyd JC. Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation. J Cell Biol 2004; 165: 653-662. https://doi.org/10.1083/jcb.200309097
  15. Masek KS, Fiore J, Leitges M, Yan SF, Freedman BD, Hunter CA. Host cell $Ca^{2+}$ and protein kinase C regulate innate recognition of Toxoplasma gondii. J Cell Sci 2006; 119(Pt 21): 4565-4573. https://doi.org/10.1242/jcs.03206
  16. Chini EN, Nagamune K, Wetzel DM, Sibley LD. Evidence that the cADPR signalling pathway controls calcium-mediated microneme secretion in Toxoplasma gondii. Biochem J 2005; 389: 269-277. https://doi.org/10.1042/BJ20041971
  17. Nagamune K, Moreno SN, Chini EN, Sibley LD. Calcium regulation and signaling in apicomplexan parasites. Subcell Biochem 2008; 47: 70-81.
  18. Bottova I, Sauder U, Olivieri V, Hehl AB, Sonda S. The P-glycoprotein inhibitor GF120918 modulates $Ca^{2+}$-dependent processes and lipid metabolism in Toxoplasma gondii. PLoS One 2010; 5: e10062. https://doi.org/10.1371/journal.pone.0010062
  19. Bennett TN, Patel J, Ferdig MT, Roepe PD. Plasmodium falciparum $Na^+$/$H^+$ exchanger activity and quinine resistance. Mol Biochem Parasitol 2007; 153: 48-58. https://doi.org/10.1016/j.molbiopara.2007.01.018
  20. Vinayak S, Alam MT, Upadhyay M, Das MK, Dev V, Singh N, Dash AP, Sharma YD. Extensive genetic diversity in the Plasmodium falciparum $Na^+$/$H^+$ exchanger 1 transporter protein implicated in quinine resistance. Antimicrob Agents Chemother 2007; 51: 4508-4511. https://doi.org/10.1128/AAC.00317-07
  21. Henry M, Briolant S, Zettor A, Pelleau S, Baragatti M, Baret E, Mosnier J, Amalvict R, Fusai T, Rogier C, Pradines B. Plasmodium falciparum $Na^+$/$H^+$ exchanger 1 transporter is involved in reduced susceptibility to quinine. Antimicrob Agents Chemother 2009; 53: 1926-1930. https://doi.org/10.1128/AAC.01243-08
  22. Andriantsoanirina V, Menard D, Rabearimanana S, Hubert V, Bouchier C, Tichit M, Bras JL, Durand R. Association of microsatellite variations of Plasmodium falciparum $Na^+$/$H^+$ exchanger (Pfnhe-1) gene with reduced in vitro susceptibility to quinine: lack of confirmation in clinical isolates from Africa. Am J Trop Med Hyg 2010; 82: 782-787. https://doi.org/10.4269/ajtmh.2010.09-0327
  23. Vercesi AE, Docampo R. Sodium-proton exchange stimulates $Ca^{2+}$ release from acidocalcisomes of Trypanosoma brucei. Biochem J 1996; 315(Pt 1): 265-270. https://doi.org/10.1042/bj3150265
  24. Vercesi AE, Rodrigues CO, Catisti R, Docampo R. Presence of a $Na^+$/$H^+$ exchanger in acidocalcisomes of Leishmania donovani and their alkalization by anti-leishmanial drugs. FEBS Lett 2000; 473: 203-206. https://doi.org/10.1016/S0014-5793(00)01531-3
  25. Mondragon R, Frixione E. $Ca^{{+}{+}}$-dependence of conoid extrusion in Toxoplasma gondii tachyzoites. J Eukaryot Microbiol 1996; 43: 120-127. https://doi.org/10.1111/j.1550-7408.1996.tb04491.x
  26. Ryning FW, Remington JS. Effect of cytochalasin D on Toxoplasma gondii cell entry. Infect Immun 1978; 20: 739-743.
  27. Silva SR, Meirelles SS, De Souza W. Mechanism of entry of Toxoplasma gondii into vertebrate cells. J Submicrosc Cytol 1982; 14: 471-482.
  28. Schwartzman JD, Pfefferkorn ER. Immunofluorescent localization of myosin at the anterior pole of the coccidian, Toxoplasma gondii. J Protozool 1983; 30: 657-661. https://doi.org/10.1111/j.1550-7408.1983.tb05339.x
  29. Dobrowolski JM, Sibley LD. Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 1996; 84: 933-939. https://doi.org/10.1016/S0092-8674(00)81071-5
  30. Cole TR, Igumenova TI. Expression and purification of the N-terminal regulatory domain of protein kinase C for biophysical studies. Protein Expr Purif 2015; 110: 14-21. https://doi.org/10.1016/j.pep.2014.12.018

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