Cloning and expression of recombinant Salmonella paratyphi A cytolethal distending toxin proteins and its effect on cytokine production by human monocyte-derived macrophages
CHEN Hong-hu1, WU Yuan-yuan2, ZHAN Li1, MEI Ling-ling1
1. Department of Microbiology, Zhejiang Provincial Center for Disease Control andPrevention, Hangzhou 310051, China; 2. Department of Clinical Laboratory, the Children's Hospital, Zhejiang University Schoolof Medicine, Hangzhou 310003, China
Abstract：To investigate the role of Salmonella paratyphi A cytolethal distending toxin (CDT) in pro-inflammatory cytokines induction in macrophages, we cloned and expressed cdtB, a subunit of CDT from Salmonella paratyphi (S. paratyphi) A in Escherichia coli, and purified the recombinant cdtB proteins (SPA-rcdtB) to homogeneity by Ni-NTA affinity chromatography. By co-cultured SPA-rcdtB with human monocyte-derived macrophages (THP-1) and detected IL-6, IL-8 and TNF-α in supernatant by ELISA, we determined if SPA-rcdtB were able to induce human monocyte-derived macrophages to produce cytokines. In addition, NF-κB inhibitor SN50 was used to research the effect of NF-κB signaling pathways in SPA-rcdtB induced macrophage IL-6, IL-8 and TNF-α secretion. In this study, we proved that the cdtb gene segment amplified from S. paratyphi A CMCC 50001 strains showed high nucleotide and amino acid sequence identities (97.62% and 99.82%) compared with cdtb gene in the GenBank (GenBank accession No. NC_006511.1). And the SPA-rcdtB were able to induce the synthesis by THP-1 of interleukin-6, IL-8 and TNF-α, and the IL-6, IL-8 and TNF-α induction by SPA-rCdtB was significantly suppressed by NF-κB inhibitor. This result indicated that SPA-rcdtB plays a promoting role inflammatory response associated with SPA infection.
陈鸿鹄, 吴圆圆, 占利, 梅玲玲. 甲型副伤寒沙门氏菌cdtB亚基的原核表达及对巨噬细胞IL-6、IL-8、TNF-α分泌的影响[J]. 中国人兽共患病学报, 2016, 32(6): 535-538.
CHEN Hong-hu, WU Yuan-yuan, ZHAN Li, MEI Ling-ling. Cloning and expression of recombinant Salmonella paratyphi A cytolethal distending toxin proteins and its effect on cytokine production by human monocyte-derived macrophages. Chinese Journal of Zoonoses, 2016, 32(6): 535-538.
[1] Wang HQ, Pan JC, Ge YM, et al. Molecular characteristics of Salmonella typhi and Salmonella paraophi a isolates in Hangzhou[J]. Chin J Microbiol Immunol, 2012, 32(8): 711-715. (in Chinese) 汪皓秋, 潘劲草, 葛玉梅, 等. 杭州地区伤寒及甲型副伤寒沙门菌流行菌株分子特征的研究[J]. 中华微生物和免疫学杂志, 2012,32(8):711-715. [2] Yan MY, Liang WL, Li W, et al. Epidemiological analysis of typhoid and paratyphoid cases in China during 1995 to 2004[J]. Dis Ctrl, 2005, 20(8): 40l-403. (in Chinese) 闫梅英, 梁未丽, 李伟, 等. 1995—2004年全国伤寒副伤寒的流行分析[J]. 疾病监测, 2005, 20(8):401-403. [3] Keuter M, Dharmana E, Gasem MH, et al. Patterns of proinflammatory cytokines and inhibitors during typhoid fever[J]. J Infect Dis, 1994, 169(6): 1306-1311. doi:10.1093/infdis/169.6.1306 [4] Jinadasa RN, Bloom SE, Weiss RS, et al. Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages[J]. Microbiology, 2011, 157(Pt 7): 1851-1875. doi:10.1099/mic.0.049536-0 [5] Haghjoo E, Galan JE. Salmonella typhi encodes a functional cytolethal distending toxin that is delivered into host cells by a bacterial-internalization pathway[J]. Proc Natl Acad Sci U S A, 2004, 101(13): 4614-4619. [6] Ando-Suguimoto ES, da Silva MP, Kawamoto D, et al. The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine production[J]. Cytokine, 2014, 66(1): 46-53. doi:10.1016/j.cyto.2013.12.014 [7] Wyant TL, Tanner MK, Sztein MB. Salmonella typhi flagella are potent inducers of proinflammatory cytokine secretion by human monocytes[J]. J Infect Dis, 1999, 67(7): 3619-3624. [8] Wang FB, Zhang XL. Type IVB pili of Salmonella typhi induce interleukin-6 expression in THP-1 cells by activating PKC signal pathway[J]. Immunol J, 2005, 21(3): 67-69. (in Chinese) 汪付兵, 章晓联. 伤寒杆菌IVB型菌毛激活PKC信号通路诱导THP-1细胞IL-6表达 [J]. 免疫学杂志, 2005, 21(3):67-69. [9] Amber KT, Bloom R, Mrowietz U, et al. TNF-alpha: a treatment target or cause of sarcoidosis[J]. J Eur Acad Dermatol, 2015, 29(11): 2104-2111. doi:10.1111/jdv.13246 [10] Nielsen AR, Pedersen BK. The biological roles of exercise-induced cytokines: IL-6, IL-8, and IL-15[J]. Appl Physiol Nutr Metab, 2007, 32(5): 833-839. doi:10.1139/H07-054 [11] Li TM, Xu H, Jiangqi XX. Research on mechanisms of mononuclear phagocyte in response to Salmonella typhi strains[J]. Chin J Public Health, 2003, 19(1): 51-52. (in Chinese) 李铁民, 徐浩, 江崎孝行.单核吞噬细胞应答伤寒沙门菌的实验研究[J]. 中国公共卫生, 2003,19(1): 51-52 [12] Idzko M, Ferrari D, Eltizschig HK. Nucleotide signalling during inflammation[J]. Nature, 2014, 509(7500): 310-317. doi:10.1038/nature13085
2016, Vol. 32 
