C型产气荚膜梭菌外毒素致小鼠肠道损伤的转录组分析

doi:10.11843/j.issn.0366-6964.2022.10.029

摘要/Abstract

摘要： 旨在探索产气荚膜梭菌外毒素造成机体炎性损伤及免疫调控紊乱的毒性机制，为产气荚膜梭菌病的致病机理研究提供理论基础。将C型产气荚膜梭菌强毒株C59-2培养上清腹腔注射BALB/c小鼠，采集小肠样本进行转录组测序，筛选差异表达基因，并对其进行GO功能注释和KEGG通路富集分析。结果显示，共获得40.99 Gb有效碱基，筛选后共得到795个差异表达基因，其中，229个基因表达上调，566个基因表达下调，对随机选取的10个基因进行q-PCR验证，其相对表达量与转录表达谱一致。GO功能注释主要涉及G蛋白偶联核苷酸受体活性和G蛋白偶联嘌呤核苷酸受体活性等。KEGG通路富集分析发现，主要富集在TNF信号通路、IL-17信号通路、p53信号通路、FOXO信号通路、Toll样受体信号通路、NF-κB信号通路等。产气荚膜梭菌外泌毒素侵入机体，会激活TNF等炎性信号通路，进而造成肠道发生炎性损伤甚至坏死。

关键词: 产气荚膜梭菌, 转录组, 肠道, 炎性信号通路

Abstract: The present study aimed to explore the toxicity mechanism of inflammatory injury and immune regulation disorders in organisms, and lay a foundation for further in-depth exploration of the pathogenic mechanism of Clostridium perfringens disease. The cultured supernatant of C. perfringens type C was intraperitoneally injected into BALB/c mice, and the small intestine samples were collected for transcriptome sequencing. Based on the differentially expressed genes (DEGs), GO functional annotation and KEGG pathway enrichment analysis were performed. The results showed that in total, there were 40.99 Gb effective bases and 795 DEGs were obtained, among which 229 were up-regulated and 566 were down-regulated. The ten randomly selected DEGs were verified by real-time fluorescent quantitative PCR that their relative expression was consistent with the transcriptional expression profile. GO functional annotation mainly involved G-protein coupled nucleotide receptor activity, and G-protein coupled purine nucleotide receptor activity. KEGG pathway enrichment analysis found that it was mainly concentrated in TNF signaling pathway, IL-17 signaling pathway, p53 signaling pathway, FOXO signaling pathway, Toll-like receptor signaling pathway, and NF-κB signaling pathway. When C. perfringens exotoxin invades the body, inflammatory signaling pathways such as TNF will be activated, resulting in inflammatory damage and even necrosis of the intestine.

Key words: Clostridium perfringens, transcriptome, intestine, inflammatory signaling pathway

中图分类号:

张思雨, 王玉炯, 曾瑾. C型产气荚膜梭菌外毒素致小鼠肠道损伤的转录组分析[J]. 畜牧兽医学报, 2022, 53(10): 3570-3581.

ZHANG Siyu, WANG Yujiong, ZENG Jin. Transcriptome Analysis of Intestinal Injury Induced by Clostridium perfringens Type C Exotoxin in Mouse[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3570-3581.

参考文献 47

[1]	FLORES-DÍAZ M, MONTURIOL-GROSS L, NAYLOR C, et al. Bacterial sphingomyelinases and phospholipases as virulence factors[J]. Microbiol Mol Biol Rev, 2016, 80(3):597-628.
[2]	ROOD J I, ADAMS V, LACEY J, et al. Expansion of the Clostridium perfringens toxin-based typing scheme[J]. Anaerobe, 2018, 53:5-10.
[3]	PETIT L, GIBERT M, POPOFF M R. Clostridium perfringens:toxinotype and genotype[J]. Trends Microbiol, 1999, 7(3):104-110.
[4]	LUO R R, HUANG X Y, YAN Z Q, et al. Identification and characterization of MAPK signaling pathway genes and associated lncRNAs in the ileum of piglets infected by Clostridium perfringens type C[J]. BioMed Res Int, 2020, 2020:8496872.
[5]	MVLLER K E, ROZGONYI F. Pathogenesis, clinical characteristics, diagnostics and treatment of bacterial foodborne diseases[J]. Orv Hetil, 2020, 161(48):2019-2028.
[6]	MARLOW M A, LUNA-GIERKE R E, GRIFFIN P M, et al. Foodborne disease outbreaks in correctional institutions-United States, 1998-2014[J]. Am J Public Health, 2017, 107(7):1150-1156.
[7]	GRASS J E, GOULD L H, MAHON B E. Epidemiology of foodborne disease outbreaks caused by Clostridium perfringens, United States, 1998-2010[J]. Foodborne Pathog Dis, 2013, 10(2):131-136.
[8]	SCALLAN E, HOEKSTRA R M, ANGULO F J, et al. Foodborne illness acquired in the United States——major pathogens[J]. Emerg Infect Dis, 2011, 17(1):7-15.
[9]	李颖, 李长青, 王彦波, 等. 一起由C型产气荚膜梭菌引起的食源性疾病致病因子检测[J]. 中国卫生检验杂志, 2016, 26(23):3379-3381.LI Y, LI C Q, WANG Y B, et al. Detection of food borne pathogenic factors caused by Clostridium perfringens[J]. Chinese Journal of Health Laboratory Technology, 2016, 26(23):3379-3381. (in Chinese)
[10]	ABD EL-HACK M E, EL-SAADONY M T, ELBESTAWY A R, et al. Necrotic enteritis in broiler chickens:disease characteristics and prevention using organic antibiotic alternatives-a comprehensive review[J]. Poult Sci, 2022, 101(2):101590.
[11]	曾瑾. 产气荚膜梭菌Beta2毒素的细胞毒性与致病机理研究[D]. 银川:宁夏大学, 2017.ZENG J. The cytotoxicity and pathogenesis of Beta2 toxin of Clostridium perfringens[D]. Yinchuan:Ningxia University, 2017. (in Chinese)
[12]	YANG Q, LIU J, WANG X F, et al. Identification of an intestinal microbiota signature associated with the severity of necrotic enteritis[J]. Front Microbiol, 2021, 12:703693.
[13]	GU C Q, LILLEHOJ H S, SUN Z F, et al. Characterization of virulent netB+/tpeL+ Clostridium perfringens strains from necrotic enteritis-affected broiler chicken farms[J]. Avian Dis, 2019, 63(3):461-467.
[14]	WADE B, KEYBURN A L, SEEMANN T, et al. Binding of Clostridium perfringens to collagen correlates with the ability to cause necrotic enteritis in chickens[J]. Vet Microbiol, 2015, 180(3-4):299-303.
[15]	刘乐文. 盲肠mRNAs与miRNAs对鸡肠炎沙门氏菌感染的表达调控[D]. 泰安:山东农业大学, 2021.LIU L W. Regulation of mRNAS and miRNAs in the response to Salmonella enterica serovar Enteritidis infection in chicken cecum[D]. Tai'an:Shandong Agricultural University, 2021. (in Chinese)
[16]	张旭, 郑敏, 吴征卓, 等. 鸭肠炎病毒感染对鸭十二指肠转录组的影响[J]. 动物医学进展, 2020, 41(10):73-79.ZHANG X, ZHENG M, WU Z Z, et al. Duodenal transcriptome sequencing at different time points after duck enteritis virus infection in ducks[J]. Progress in Veterinary Medicine, 2020, 41(10):73-79. (in Chinese)
[17]	姜天团. C型产气荚膜梭菌致仔猪腹泻的蛋白质组学研究及其与转录组数据的联合分析[D]. 兰州:甘肃农业大学, 2019.JIANG T T. Proteomics study of piglet diarrhea caused by Clostridium perfringens type C and its combined analysis with transcriptome data[D]. Lanzhou:Gansu Agricultural University, 2019. (in Chinese)
[18]	FERNANDEZ-MIYAKAWA M E, MARCELLINO R, UZAL F A. Clostridium perfringens type A toxin production in 3 commonly used culture media[J]. J Vet Diagn Invest, 2007, 19(2):184-186.
[19]	MA M L, VIDAL J, SAPUTO J, et al. The VirS/VirR two-component system regulates the anaerobic cytotoxicity, intestinal pathogenicity, and enterotoxemic lethality of Clostridium perfringens type C isolate CN3685[J]. mBio, 2011, 2(1):e00338-10.
[20]	房晓文. 內蒙羊猝狙及羊快疫病原诊断报告[J]. 畜牧兽医学报, 1956, 1(1):19-31.FANG X W. Studies on "struck" and "braxy" in sheep and goats in Inner Mongolia[J]. Acta Veterinaria Et Zootechnica Sinica, 1956, 1(1):19-31. (in Chinese)
[21]	FISHER D J, FERNANDEZ-MIYAKAWA M E, SAYEED S, et al. Dissecting the contributions of Clostridium perfringens type C toxins to lethality in the mouse intravenous injection model[J]. Infect Immun, 2006, 74(9):5200-5210.
[22]	MORTAZAVI A, WILLIAMS B A, MCCUE K, et al. Mapping and quantifying mammalian transcriptomes by RNA-Seq[J]. Nat Methods, 2008, 5(7):621-628.
[23]	LACHMANN A, CLARKE D J B, TORRE D, et al. Interoperable RNA-Seq analysis in the cloud[J]. Biochim Biophys Acta Gene Regul Mech, 2020, 1863(6):194521.
[24]	LIAO Y, SMYTH G K, SHI W. Feature Counts:an efficient general purpose program for assigning sequence reads to genomic features[J]. Bioinformatics, 2014, 30(7):923-930.
[25]	LOVE M I, HUBER W, ANDERS S. Moderated estimation of fold change and dispersion for RNA-Seq data with DESeq2[J]. Genome Biol, 2014, 15(12):550.
[26]	The Gene Ontology Consortium. The gene ontology resource:20 years and still GOing strong[J]. Nucl Acids Res, 2019, 47(D1):D330-D338.
[27]	YOUNG M D, WAKEFIELD M J, SMYTH G K, et al. Gene ontology analysis for RNA-Seq:accounting for selection bias[J]. Genome Biol, 2010, 11(2):R14.
[28]	KANEHISA M, FURUMICHI M, TANABE M, et al. KEGG:new perspectives on genomes, pathways, diseases and drugs[J]. Nucl Acids Res, 2017, 45(D1):D353-D361.
[29]	MARKS S L, KATHER E J. Bacterial-associated diarrhea in the dog:a critical appraisal[J]. Vet Clin North Am:Small Anim Pract, 2003, 33(5):1029-1060.
[30]	ZUO T, KAMM M A, COLOMBEL J F, et al. Urbanization and the gut microbiota in health and inflammatory bowel disease[J]. Nat Rev Gastroenterol Hepatol, 2018, 15(7):440-452.
[31]	NELL S, SUERBAUM S, JOSENHANS C. The impact of the microbiota on the pathogenesis of IBD:lessons from mouse infection models[J]. Nat Rev Microbiol, 2010, 8(8):564-577.
[32]	GUAN Q D. A comprehensive review and update on the pathogenesis of inflammatory bowel disease[J]. J Immunol Res, 2019, 2019:7247238.
[33]	ROOS S, WYDER M, CANDI A, et al. Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium[J]. Toxins, 2015, 7(4):1235-1252.
[34]	THIEL A, MOGEL H, BRUGGISSER J, et al. Effect of Clostridium perfringens β-toxin on platelets[J]. Toxins (Basel), 2017, 9(10):336.
[35]	AUTHEMAN D, WYDER M, POPOFF M, et al. Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells[J]. PLoS One, 2013, 8(5):e64644.
[36]	NAGAHAMA M, SHIBUTANI M, SEIKE S, et al. The p38 MAPK and JNK pathways protect host cells against Clostridium perfringens beta-toxin[J]. Infect Immun, 2013, 81(10):3703-3708.
[37]	SOLANKI A K, PANWAR D, KAUSHIK H, et al. Molecular docking analysis of P2X7 receptor with the beta toxin from Clostridium perfringens[J]. Bioinformation, 2020, 16(8):594-601.
[38]	SUN X M, ZHOU R X, LEI Y P, et al. The ligand-gated ion channel P2X7 receptor mediates NLRP3/caspase-1-mediated pyroptosis in cerebral cortical neurons of juvenile rats with sepsis[J]. Brain Res, 2020, 1748:147109.
[39]	ZHAO S H, ZHOU Y H, FAN Y, et al. Involvement of purinergic 2X4 receptor in glycoprotein 120-induced pyroptosis in dorsal root ganglia[J]. J Neurochem, 2019, 151(5):584-594.
[40]	YUAN Y H, DING D K, ZHANG N, et al. TNF-α induces autophagy through ERK1/2 pathway to regulate apoptosis in neonatal necrotizing enterocolitis model cells IEC-6[J]. Cell Cycle, 2018, 17(11):1390-1402.
[41]	BRADLEY J R. TNF-mediated inflammatory disease[J]. J Pathol, 2008, 214(2):149-160.
[42]	SUCHODOLSKI J S. Companion animals symposium:microbes and gastrointestinal health of dogs and cats[J]. J Anim Sci, 2011, 89(5):1520-1530.
[43]	HU X R, ZHANG K, CHEN Z Q, et al. The HMGB1-IL-17A axis contributes to hypoxia/reoxygenation injury via regulation of cardiomyocyte apoptosis and autophagy[J]. Mol Med Rep, 2018, 17(1):336-341.
[44]	MCGEACHY M J, CUA D J, GAFFEN S L. The IL-17 family of cytokines in health and disease[J]. Immunity, 2019, 50(4):892-906.
[45]	LI L L, DAI B, SUN Y H, et al. The activation of IL-17 signaling pathway promotes pyroptosis in pneumonia-induced sepsis[J]. Ann Transl Med, 2020, 8(11):674.
[46]	LEI-LESTON A C, MURPHY A G, MALOY K J. Epithelial cell inflammasomes in intestinal immunity and inflammation[J]. Front Immunol, 2017, 8:1168.
[47]	OZAKI E, CAMPBELL M, DOYLE S L. Targeting the NLRP3 inflammasome in chronic inflammatory diseases:current perspectives[J]. J Inflamm Res, 2015, 8:15-27.