布鲁氏菌Ⅳ型分泌系统效应蛋白VceC对山羊滋养层细胞内质网应激和性腺激素分泌的影响

doi:10.11843/j.issn.0366-6964.2023.03.032

摘要/Abstract

摘要： 旨在研究布鲁氏菌IV型分泌系统效应分子VceC对山羊滋养层细胞(GTC)内质网应激及性腺激素分泌的影响，为揭示其在布鲁氏菌感染宿主细胞中的作用，阐明布鲁氏菌胞内生存和引起动物流产的机制提供重要依据。本研究通过构建VceC的真核表达载体pEGFP-C1-VceC并转染GTC，Western blot检测内质网应激标志性分子GRP78和CHOP蛋白表达量变化，qRT-PCR和Western blot进一步检测未折叠蛋白反应(UPR)信号通路相关分子；ELISA检测细胞培养上清液的孕酮和雌激素的浓度变化。结果表明，成功构建了VceC真核表达载体pEGFP-C1-VceC，转染GTC后，12和24 h GRP78蛋白表达均显著升高(P<0.01)，24 h后CHOP蛋白表达显著降低(P<0.05)；qRT-PCR检测IRE1和XBP-1的mRNA表达量在24 h均显著升高(P<0.05)，而PERK、ATF6和ATF4 mRNA的表达未发生显著变化(P>0.05)，Western blot检测IRE1蛋白的表达在转染12 h后显著升高(P<0.01)；VceC转染组细胞培养上清液中孕酮含量在转染12和24 h后显著低于空白组和空载体组(P<0.01)，雌激素无显著差异(P>0.05)。证明布鲁氏菌VceC不仅能通过激活UPR中的IRE1通路从而激发GTC的内质网应激反应，还能改变GTC中孕酮/雌激素的比值。

关键词: 布鲁氏菌, IV型分泌系统, VceC效应蛋白, 山羊滋养层细胞, 内质网应激, 性腺激素

Abstract: We aimed to study the effects of Brucella Type IV secretes system effector protein VceC on endoplasmic reticulum stress and gonadal hormone secretory in goat trophoblast cells, and to reveal its role in Brucella infection of host cells, which is of great significance for clarifying the mechanism of intracellular survival of Brucella and miscarriage in animals. In this experiment the eukaryotic expression vector pEGFP-C1-VceC of VceC was constructed and transfected it into GTC, then the main factors of ERS, GRP78 and CHOP, were detected by Western blot method. Then the related molecules of unfolded protein reaction (UPR) were further detected by qRT-PCR and Western blot methods. The concentration of Progesterone and Estrogen secreted by GTC were detected by ELISA. The results showed that the VceC eukaryotic expression vector pEGFP-C1-VceC was constructed and transfected it into GTC successfully. The protein expression of main factors of ERS, GRP78 were significantly increased (P<0.01) at 12 and 24 h after transfection of pEGFP-C1-VceC into GTC, while CHOP protein expression was significantly decreased (P<0.05) at 24 h after transfection. According to the qRT-PCR results, the mRNA expression levels of IRE1 and XBP-1 were significantly increased (P<0.05) at 24 h after transfection of pEGFP-C1-VceC, while PERK, ATF6 and ATF4 mRNA had little change (P>0.05). According to the Western blot results, the protein expression of IRE1 was significantly increased at 12 h after transfection of pEGFP-C1-VceC (P<0.01). In addition, the content of progesterone in cell culture supernatant of the VceC transfection group decreased significantly (P<0.01) at 12 and 24 h than those in the GTC group and pEGFP-C1 transfected GTC group, while the content of Estrogen had little change (P>0.05). The results suggest that VceC can activate the ERS of GTC by activating the IRE1 pathway of unfolded protein response. Moreover, VceC protein also changes the Progesterone/Estrogen ratio in GTC.

Key words: Brucella, type IV secretion system, VceC effector protein, goat trophoblastic cells, endoplasmic reticulum stress, gonadal hormone

中图分类号:

S855.12

相彩霞, 王相国, 李俊玫, 支飞杰, 房姣阳, 郑维芳, 陈家露, 靳亚平, 王爱华. 布鲁氏菌Ⅳ型分泌系统效应蛋白VceC对山羊滋养层细胞内质网应激和性腺激素分泌的影响[J]. 畜牧兽医学报, 2023, 54(3): 1210-1220.

XIANG Caixia, WANG Xiangguo, LI Junmei, ZHI Feijie, FANG Jiaoyang, ZHENG Weifang, CHEN Jialu, JIN Yaping, WANG Aihua. The Influence of Brucella Type IV Secretes System Effector Protein VceC on Endoplasmic Reticulum Stress and Gonadal Hormone Secretory in Goat Trophoblast Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1210-1220.

参考文献 38

[1]	陆承平.兽医微生物学[M]. 5版.北京:中国农业出版社, 2013.LU C P. Veterinary Microbiology[M]. 5th Edition. Beijing:China Agriculture Press, 2013.(in Chinese)
[2]	ATLURI V L, XAVIER M N, DE JONG M F, et al. Interactions of the human pathogenic Brucella species with their hosts[J]. Annu Rev Microbiol, 2011, 65:523-541.
[3]	CASWELL C C. Assessment of survival and replication of Brucella spp. in murine peritoneal macrophages[M]//ALLEN I C. Mouse Models of Innate Immunity. New York:Humana Press, 2019, 1960:181-189.
[4]	FRANCO M P, MULDER M, SMITS H L. Persistence and relapse in brucellosis and need for improved treatment[J]. Trans R Soc Trop Med Hyg, 2007, 101(9):854-855.
[5]	BYNDLOSS M X, TSAI A Y, WALKER G T, et al. Brucella abortus infection of placental trophoblasts triggers endoplasmic reticulum stress-mediated cell death and fetal loss via type IV secretion system-dependent activation of CHOP[J]. mBio, 2019, 10(4):e01538-19.
[6]	ZHOU Y C, BU Z Y, QIAN J, et al. The UTP-glucose-1-phosphate uridylyltransferase of Brucella melitensis inhibits the activation of NF-κB via regulating the bacterial type IV secretion system[J]. Int J Biol Macromol, 2020, 164:3098-3104.
[7]	DE JONG M F, SUN Y H, DEN HARTIGH A B, et al. Identification of VceA and VceC, two members of the VjbR regulon that are translocated into macrophages by the Brucella type IV secretion system[J]. Mol Microbiol, 2008, 70(6):1378-1396.
[8]	DE JONG M F, STARR T, WINTER M G, et al. Sensing of bacterial type IV secretion via the unfolded protein response[J]. mBio, 2013, 4(1):e00418-12.
[9]	ZIMMERMANN R. Components and mechanisms of import, modification, folding, and assembly of immunoglobulins in the endoplasmic reticulum[J]. J Clin Immunol, 2016, 36 Suppl 1:5-11.
[10]	WANG M, KAUFMAN R J. Protein misfolding in the endoplasmic reticulum as a conduit to human disease[J]. Nature, 2016, 529(7586):326-335.
[11]	PLUQUET O, POURTIER A, ABBADIE C. The unfolded protein response and cellular senescence. A review in the theme:cellular mechanisms of endoplasmic reticulum stress signaling in health and disease[J]. Am J Physiol Cell Physiol, 2015, 308(6):C415-C425.
[12]	GORMAN A M, HEALY S J M, JÄGER R, et al. Stress management at the ER:regulators of ER stress-induced apoptosis[J]. Pharmacol Ther, 2012, 134(3):306-316.
[13]	CLÁUDIO N, DALET A, GATTI E, et al. Mapping the crossroads of immune activation and cellular stress response pathways[J]. EMBO J, 2013, 32(9):1214-1224.
[14]	TODD D J, LEE A H, GLIMCHER L H. The endoplasmic reticulum stress response in immunity and autoimmunity[J]. Nat Rev Immunol, 2008, 8(9):663-674.
[15]	MCCONKEY C A, DELORME-AXFORD E, NICKERSON C A, et al. A three-dimensional culture system recapitulates placental syncytiotrophoblast development and microbial resistance[J]. Sci Adv, 2016, 2(3):e1501462.
[16]	SFERRUZZI-PERRI A N, CAMM E J. The programming power of the placenta[J]. Front Physiol, 2016, 7:33.
[17]	MARTÍNEZ R, DUNNER S, TORO R, et al. Effect of polymorphisms in the Slc11a1 coding region on resistance to brucellosis by macrophages in vitro and after challenge in two Bos breeds (Blanco Orejinegro and Zebu)[J]. Genet Mol Biol, 2010, 33(3):463-470.
[18]	BOSCHIROLI M L, OUAHRANI-BETTACHE S, FOULONGNE V, et al. The Brucella suis virB operon is induced intracellularly in macrophages[J]. Proc Natl Acad Sci U S A, 2002, 99(3):1544-1549.
[19]	CELLI J, DE CHASTELLIER C, FRANCHINI D M, et al. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum[J]. J Exp Med, 2003, 198(4):545-556.
[20]	SALCEDO S P, MARCHESINI M I, DEGOS C, et al. BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions[J]. Front Cell Infect Microbiol, 2013, 3:28.
[21]	SENGUPTA D, KOBLANSKY A, GAINES J, et al. Subversion of innate immune responses by Brucella through the targeted degradation of the TLR signaling adapter, MAL[J]. J Immunol, 2010, 184(2):956-964.
[22]	MYENI S, CHILD R, NG T W, et al. Brucella modulates secretory trafficking via multiple type IV secretion effector proteins[J]. PLoS Pathog, 2013, 9(8):e1003556.
[23]	DÖHMER P H, VALGUARNERA E, CZIBENER C, et al. Identification of a type IV secretion substrate of Brucella abortus that participates in the early stages of intracellular survival[J]. Cell Microbiol, 2014, 16(3):396-410.
[24]	DUDEK J, BENEDIX J, CAPPEL S, et al. Functions and pathologies of BiP and its interaction partners[J]. Cell Mol Life Sci, 2009, 66(9):1556-1569.
[25]	MUNRO S, PELHAM H R B. An Hsp70-like protein in the ER:identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein[J]. Cell, 1986, 46(2):291-300.
[26]	GIORGI C, BONORA M, SORRENTINO G, et al. p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner[J]. Proc Natl Acad Sci U S A, 2015, 112(6):1779-1784.
[27]	OYADOMARI S, MORI M. Roles of CHOP/GADD153 in endoplasmic reticulum stress[J]. Cell Death Differ, 2004, 11(4):381-389.
[28]	QIN Q M, PEI J W, ANCONA V, et al. RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1α in supporting Brucella replication[J]. PLoS Pathog, 2008, 4(7):e1000110.
[29]	SMITH J A, KHAN M, MAGNANI D D, et al. Brucella induces an unfolded protein response via TcpB that supports intracellular replication in macrophages[J]. PLoS Pathog, 2013, 9(12):e1003785.
[30]	TAGUCHI Y, IMAOKA K, KATAOKA M, et al. Yip1A, a novel host factor for the activation of the IRE1 pathway of the unfolded protein response during Brucella infection[J]. PLoS Pathog, 2015, 11(3):e1004747.
[31]	HE Y Q, REICHOW S, RAMAMOORTHY S, et al. Brucella melitensis triggers time-dependent modulation of apoptosis and down-regulation of mitochondrion-associated gene expression in mouse macrophages[J]. Infect Immun, 2006, 74(9):5035-5046.
[32]	TOLOMEO M, DI CARLO P, ABBADESSA V, et al. Monocyte and lymphocyte apoptosis resistance in acute and chronic brucellosis and its possible implications in clinical management[J]. Clin Infect Dis, 2003, 36(12):1533-1538.
[33]	CHEN F, HE Y Q. Caspase-2 mediated apoptotic and necrotic murine macrophage cell death induced by rough Brucella abortus[J]. PLoS One, 2009, 4(8):e6830.
[34]	JI L, BRKIĆ J, LIU M, et al. Placental trophoblast cell differentiation:physiological regulation and pathological relevance to preeclampsia[J]. Mol Aspects Med, 2013, 34(5):981-1023.
[35]	HALASZ M, SZEKERES-BARTHO J. The role of progesterone in implantation and trophoblast invasion[J]. J Reprod Immunol, 2013, 97(1):43-50.
[36]	WANG X G, LIN P F, LI Y, et al. Brucella suis vaccine strain 2 induces endoplasmic reticulum stress that affects intracellular replication in goat trophoblast cells in vitro[J]. Front Cell Infect Microbiol, 2016, 6:19.
[37]	王相国.内质网应激在布鲁氏菌体外感染山羊子宫内膜与滋养层细胞中的作用[D].杨凌:西北农林科技大学, 2016.WANG X G. The Role of endoplasmic reticulum stress in Brucella-infected goat endometrial and trophoblast cells in vitro[D].Yangling:Northwest A&F University, 2016.(in Chinese)
[38]	MARTIROSYAN A, PÉREZ-GUTIERREZ C, BANCHEREAU R, et al. Brucella β 1, 2 cyclic Glucan is an activator of human and mouse dendritic cells[J]. PLoS Pathog, 2012, 8(11):e1002983.