Expression and localization of the spore wall protein SWP26 of Nosema bombycis in the silkworm BmN cell line

Feng Zhu, Zhongyuan Shen, Shengyan Xiao, Yajie Yue, Xuliang Fu, Xudong Tang, Li Xu, Xijie Guo

Jiangsu University of Science and Technology, Zhenjiang, China.
Jiangsu University of Science and Technology, Zhenjiang, China Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China.

DOI: 10.4236/as.2013.42013                

Abstract

The microsporidian spore wall proteins, as the main components of the spore wall, play a key role in spore adherence to host cells and in recognition of the parasite by the host during the invasion process. In this study, we used the Bac-to-Bac baculovirus expression system to express the spore wall protein SWP26, fused to enhanced green fluorescent protein (EGFP), in the silkworm BmN cell line. The SWP26 and EGFP genes were inserted into the baculovirus transfer vector pFastBac1. The transfer vector pFastBac1-swp26-egfp was transformed into the bacterium Escherichia coli DHl0Bac/Bombyx mori nucleopolyhedrovirus (BmNPV) to construct the recombinant vBm^swp26-egfp bacmid. The vBm^swp26-egfp bacmid DNA was then used to transfect BmN cells to obtain the recombinant baculovirus. Western blotting analysis of total protein lysates in BmN cells infected by the recombinant virus showed a protein band of approximately 51 kDa, which corresponded to the deduced molecular weight of the swp26-egfp fusion protein. In addition, a fluorescence signal was observed in the cytoplasm and nucleoplasm of transfected cells, indicating that SWP26 had been successfully expressed in BmN cells. The SWP26 expression system established in this study lays the foundation for additional molecular and cellular studies, especially those focused on the interaction between the SWP26 protein of Nosema bombycis and the proteins of the silkworm, Bombyx mori.

Keywords

Bacmid; Expression; Microsporidia; Nosema bombycis; Spore Wall Protein

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Wittner, M. and Weiss, L.M. (1999) The microsporidia and microsporidiosis. ASM Press, Washington DC.
[2]	Becnel, J.J. and Andreadis, T.G. (1999) Microsporidia in insects. In: Wittner, M. and Weiss, L.M. Eds., The Mi crosporidia and Microsporidiosis, ASM Press, Washing ton DC, 447-501.
[3]	Tanada, Y. and Kaya, H.K. (1993) Insect pathology. Aca demic Press, New York.
[4]	Vávra, J. and Larsson, J.I.R. (1999) Structure of the mi crosporidia. In: Wittner, M. and Weiss, L.M., Eds., The Microsporidia and Microsporidiosis, ASM Press, Wash ington, DC, 7-84.
[5]	Southern, T.R., Jolly, C.E., Lester, M.E. and Hayman, J.R. (2007) EnP1, a microsporidian spore wall protein that enables spores to adhere to and infect host cells in vitro. Eukaryotic Cell, 6, 1354-1362. doi:10.1128/EC.00113-07
[6]	Brosson, D., Kuhn, L., Delbac, F., Garin, J., Vivares, C.P. and Texier, C. (2006) Proteomic analysis of the eu karyotic parasite Encephalitozoon cuniculi (microspo ridia): A reference map for proteins expressed in late sporogonial stages. Proteomics, 6, 3625-3635. doi:10.1002/pmic.200500796
[7]	Bohne, W., Ferguson, D.J., Kohler, K. and Gross, U. (2000) Developmental expression of a tandemly repeated, glycine and serine-rich spore wall protein in the micro sporidian pathogen Encephalitozoon cuniculi. Infection and Immunity, 68, 2268-2275. doi:10.1128/IAI.68.4.2268-2275.2000
[8]	Hayman, J.R., Hayes, S.F., Amon, J. and Nash, T.E. (2001) Developmental expression of two spore wall proteins during maturation of the microsporidian Encephalitozoon intestinalis. Infection and Immunity, 69, 7057-7066. doi:10.1128/IAI.69.11.7057-7066.2001
[9]	Brosson, D., Kuhn, L., Prensier, G., Vivares, C.P. and Texier, C. (2005) The putative chitin deacetylase of Encephalitozoon cuniculi: A surface protein implicated in microsporidian spore-wall formation. FEMS Microbiol ogy Letters, 247, 81-90. doi:10.1016/j.femsle.2005.04.031
[10]	Peuvel-Fanget, I., Polonais, V., Brosson, D., Texier, C., Kuhn, L., Peyret, P., Vivares, C. and Delbac, F. (2006) EnP1 and EnP2, two proteins associated with the En cephalitozoon cuniculi endospore, the chitin-rich inner layer of the microsporidian spore wall. International Jour nal for Parasitology, 36, 309-318. doi:10.1016/j.ijpara.2005.10.005
[11]	Xu, Y., Takvorian, P., Cali, A., Wang, F., Zhang, H., Orr, G. and Weiss, L.M. (2006) Identification of a new spore wall protein from Encephalitozoon cuniculi. Infection and Immunity, 74, 239-247. doi:10.1128/IAI.74.1.239-247.2006
[12]	Cai, S., Lu, X., Qiu, H., Li, M. and Feng, Z. (2011) Iden tification of a Nosema bombycis (Microsporidia) spore wall protein corresponding to spore phagocytosis. Para sitology, 138, 1102-1109. doi:10.1017/S0031182011000801
[13]	Li, Y., Wu, Z., Pan, G., He, W., Zhang, R., Hu, J. and Zhou, Z. (2009) Identification of a novel spore wall pro tein (SWP26) from microsporidia Nosema bombycis. International Journal for Parasitology, 39, 391-398.
[14]	Wu, Z., Li, Y., Pan, G., Tan, X., Hu, J., Zhou, Z. and Xiang, Z. (2008) Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis (Microsporidia). Proteomics, 8, 2447-2461. doi:10.1002/pmic.200700584
[15]	Wu, Z., Li, Y., Pan, G., Zhou, Z. and Xiang, Z. (2009) SWP25, a novel protein associated with the Nosema bombycis endospore. Journal of Eukaryotic Microbiology, 56, 113-118. doi:10.1111/j.1550-7408.2008.00375.x
[16]	Li, Z., Pan, G., Li, T., Huang, W., Chen, J., Geng, L., Yang, D., Wang, L. and Zhou, Z. (2012) SWP5, a spore wall protein, interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis. Eukaryotic Cell, 11, 229-237. doi:10.1128/EC.05127-11
[17]	Dong, S., Shen, Z., Xu, L. and Zhu, F. (2009) Sequence and phylogenetic analysis of SSU rRNA gene of five mi crosporidia. Current Microbiology, 60, 30-37. doi:10.1007/s00284-009-9495-7
[18]	Hayman, J.R., Southern, T.R. and Nash, T.E. (2005) Role of sulfated glycans in adherence of the microsporidian Encephalitozoon intestinalis to host cells in vitro. Infec tion and Immunity, 73, 841-848. doi:10.1128/IAI.73.2.841-848.2005
[19]	Cali, A. and Takvorian, P.M. (1999) Developmental mor phology and life cycles of the microsporidia. In: Wittner, M. and Weiss, L. Eds., The Microsporidia and Micro sporidiosis, American Society of Microbiology, Washing ton DC, 85-128.
[20]	Enriquez, F.J., Wagner, G., Fragoso, M. and Ditrich, O. (1998) Effects of an anti-exospore monoclonal antibody on microsporidial development in vitro. Parasitology, 117, 515-520. doi:10.1017/S0031182098003345
[21]	Sak, B., Sakova, K. and Ditrich, O. (2004) Effects of a novel anti-exospore monoclonal antibody on microspori dial development in vitro. Parasitology Research, 92, 74-80. doi:10.1007/s00436-003-0988-1
[22]	Zhang, F., Lu, X., Kumar, V.S., Zhu, H., Chen, H., Chen, Z. and Hong, J. (2007) Effects of a novel anti-exospore monoclonal antibody on microsporidial Nosema bomby cis germination and reproduction in vitro. Parasitology, 134, 1551-1558. doi:10.1017/S0031182007002934
[23]	Motohashi, T., Shimojima, T., Fukagawa, T., Maenaka, K. and Park, E.Y. (2005) Efficient large-scale protein pro duction of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system. Biochemical and Biophysical Research Communications, 326, 564-569. doi:10.1016/j.bbrc.2004.11.060