Please wait a minute...
Journal of ZheJiang University(Medical Science)  2013, Vol. 42 Issue (2): 141-148    DOI: 10.3785/j.issn.1008-9292.2013.02.003
    
Role of fliY gene in pathogenicity-associated chemotaxis and colonization of Campylobacter jejuni
LOU Hong-Qiang1, GE Yu-Mei2, ZHANG Jin-Liang1, YAN Jie2, ZHAO Jin-Fang3
1.Medical School of Jinhua Occupational Technique College,Jinhua 321007,China; 2.Department of Medical Microbiology and Parasitology,Zhejiang University School of Medicine,Hangzhou 310058,China; 3.Department of Laboratory Medicine,The First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou 310006,China;
Download: HTML (   PDF(1541KB)
Export: BibTeX | EndNote (RIS)      

Abstract  

Objective: To construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility,chemotaxis and colonization.
Methods: The plasmid pBluescript-Ⅱ-SK was used to construct the suicide plasmid; according to homologous exchange principle,the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168.The fliY mutant strain was identified by PCR,sequencing and Western blotting.The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice.
Results: The fliY- mutant strain showed a growth curve in medium similar to that of wild-type strain.PCR,sequencing and Western blotting assay confirmed that the fliY gene in fliY- mutant was deleted.Compared to the wild-type strain,the colonies of fliY- mutant on semisolid plate were much smaller(P<0.05),the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated(P<0.05),and the number of fliY mutant(CFU) in jejunal tissue specimens of the infected mice was significantly decreased(P<0.05).
Conclusion: The function of C.jejuni fliY gene refers to controlling flagellar movement,which is involved in bacterial chemotaxis and colonization.



Key wordsCampylobacter jejuni/pathogenicity      Mice,knockout      Flagella      Polymerase chain reaction      Blotting,Western      Flagellar motor switch protein      Motility      Chemotaxis      Colonization     
Received: 28 December 2012      Published: 25 March 2013
CLC:  R 378.2  
Cite this article:

LOU Hong-Qiang, GE Yu-Mei, ZHANG Jin-Liang, YAN Jie, ZHAO Jin-Fang. Role of fliY gene in pathogenicity-associated chemotaxis and colonization of Campylobacter jejuni. Journal of ZheJiang University(Medical Science), 2013, 42(2): 141-148.

URL:

http://www.zjujournals.com/med/10.3785/j.issn.1008-9292.2013.02.003     OR     http://www.zjujournals.com/med/Y2013/V42/I2/141


fliY基因在空肠弯曲菌致病性相关趋化和定植中的作用

目的:构建空肠弯曲菌(Campylobacter jejuni)鞭毛马达开关蛋白FliY编码基因(fliY)敲除突变株,了解FliY蛋白控制细菌鞭毛运动的作用及其与细菌趋化和定植的关系。
方法:采用pBluescript-Ⅱ-SK质粒构建用于敲除空肠弯曲菌NCTC11168株fliY基因的自杀质粒。根据同源交换原理,利用自杀质粒构建空肠弯曲菌fliY基因敲除突变株(fliY-)。采用PCR、PCR产物测序与Western Blot,对fliY-突变株进行鉴定。采用体外菌落迁徙试验、细菌趋化试验以及小鼠空肠定植试验,检测fliY-突变株趋化和定植能力。
结果:fliY-突变株生长曲线与野生株相似,但PCR与测序及Western Blot结果显示,fliY--突变株中fliY基因已被敲除。与野生株比较,fliY-突变株半固体平板上菌落明显较小(P<0.05),对脱氧胆酸钠和牛胆汁趋化能力显著下降(P<0.05),在感染小鼠空肠组织标本中的细菌数(CFU)也明显减少(P<0.05)。
结论:空肠弯曲菌fliY基因功能与调控鞭毛运动作用有关,从而对细菌趋化和定植过程产生重大影响。


关键词: 弯曲杆菌,  空肠/致病力,  小鼠,  基因敲除,  鞭毛,  聚合酶链反应,  印迹法,  蛋白质,  鞭毛马达开关蛋白,  运动力,  趋化,  定植 

[1]ALLOS B M.Campylobacter jejuni infections: update on emerging issues and trends
[J].Clin Infect Dis,2001,32(8):1201-1206.

[2]ZIBAUER M,DORRELL N,WREN B W,et al.Campylobacter jejuni-mediated disease pathogensis: an update
[J].Trans R Soc Trop Med Hyg,2008,102(2): 123-129.

[3]Van DOORN PA,RUTS L,JACOBS BC.Clinical features,pathogenesis,and treatment of Guillain-Barr syndrome
[J].Lancet Neurol,2008,7(10): 939-950.

[4]SZURMANT H,ORDAL GW.Diversity in chemotaxis mechanisms among bacteria and archaea
[J].Microbiol Mol Biol Rev,2004,68(2):301-309.

[5]TERRY K,WILLIAMS S M,CONNOLLY L,et al.Chemotaxis plays multiple roles during Helicobacter pylori animal infection
[J].Infect Immun,2005,73(2):803-811.

[6]MARICONDA S,WANG Q,HARSHEY R M.A mechanical role for the chemotaxis system in swarming motility
[J].Mol Microbiol,2006,60(6):1590-1602.

[7]YOUNG K T,DAVIS L M,DIRITA V J.Campylobacter jejuni: molecular biology and pathogenesis.Nature Rev Microbiol,2007,5(9):665-679.

[8]LI Zhifeng,ZHAO Jinfang,LOU Hongqiang,et al(李志峰,赵金方,楼宏强,等).Prokaryotic expression of Campylobacter jejuni mcp1/2/3 genes and relationship among the expressed products and bacterial chemotactic behavior
[J].Chinese Journal of Microbiology and Immunology(中华微生物学和免疫学杂志),2009,29(7):607-611.(in Chinese)

[9]LOU Hongqiang,LIAO Shumei,HU Ye,et al(楼宏强,廖苏梅,胡野,等).Targeting knock out of Leptospira interrogans flagellum-associated fliN gene and pathogenic function alteration of the mutant
[J].Chinese Journal of Microbiology and Immunology(中华微生物学和免疫学杂志),2009,29(8):677-682.(in Chinese)

[10]LIAO S M,SUN A H,OJCIUS D M,et al.Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai
[J].BMC Microbiol,2009,9:253-263.

[11]SENESI S,CELANDRONI F,SALVETTI S,et al.Swarming motility in Bacillus cereus and characterization of a fliY mutant impaired in swarm cell differentiation
[J].Microbiology,2002,148(Pt6):1785-1794.

[12]RAJAGOPALA S V,TITZ B,GOLL J,et al.The protein network of bacterial motility
[J].Mol Syst Biol,2007,3:128.

[13]PARKHILL J,WREN B W,MUNQALL K,et al.The genome sequence of food-borne pathogen Campylobacter jejuni reveals hypervariable sequence
[J].Nature,2000,403(6770):665-668.

[14]SAMBROOK J,FRITSCH E F,MANIATIS T.Molecular cloning,a latoratory manual
[M].New York: Cold Spring Harbor Laboratory Press,1989,1.21-1.52,2.60-2.80,7.30-7.35,9.14-9.22.

[15]WASSENAAR T M,FRY B N,van der ZEIJST B A.Genetic manipulation of Campylobacter: evaluation of natural transformation and electro-transformation
[J].Gene, 1993,132(1):131-135.

[16]HINDS J,MAHENTHIRALINGAM E,KEMPSELL K E, et al.Enhanced gene replacement in mycobacteria
[J].Microbiology,1999,145(Pt 3):519-527.

[17]BRONDSTED L,ANDERSEN M T,PARKER M,et al.The HtrA protease of Campylobacter jejuni is required for heat and oxygen tolerance and for optimal interaction with human epithelial cells
[J].Appl Environ Microbiol,2005,71(6):3205-3212.

[18]RUAN Ping,SUN Aihua,ZHAO Xing,et al(阮萍,孙爱华,赵欣,等).Contribution of motA gene in pathogenesis-associated chemotaxis and colonization of Campylobacter jejuni
[J].Chinese Journal of Microbiology and Immunology(中华微生物学和免疫学杂志),2010,30(3):195-200.(in Chinese)

[19]PEI Z,BURUCOA C,GRIGNON B,et al.Mutation in the peb1A locus of Campylobacter jejuni reduces interactions with epithelial cells and intestinal colonization of mice
[J].Infect Immun,1998,66(3):938-943.

[20]KORETKE K K,LUPAS A N,WARREN P V.Evolution of two-component signal transduction
[J].Mol Biol Evol,2000,17(12):1956-1970.

[21]WANG Yuan,LOU Hongqiang,WANG Huan,et al(王媛,楼宏强,王欢,等).CheA/CheY signaling system responsible for chemotaxis in vitro and colonization in vivo of Campylobacter jejuni 
[J].Chinese Journal of Microbiology and Immunology(中华微生物学和免疫学杂志),2011,31(3):201-207.(in Chinese)

[22]KIRSTICH C J,ORDAL G W.Analysis of chimeric chemoreceptor in Bacillus subtilis reveals a role for CheD in the function of MepC HAMP domain
[J].J Bacteriol,2004,186(17): 5950-5955.

[23]HOSKING E R,MANSON M D.Clusters of charged residues at the C terminus of MotA and N terminus of MotB are important for function of the Escherichia coli flagellar motor
[J].J Bacteriol,2008,190(15):5517-5521.

[24]LOWENTHAL A C,HILL M,SYCURO L K,et al.Functional analysis of the Helicobacter pylori flagellar switch proteins
[J].J Bacteriol,2009,191(23):7147-7156.

[25]BROWN P N,TERRAZAS M,PAUL K,et al. Mutational analysis of the flagellar protein FliG: sites of interaction with FliM and implications for organization of the switch complex
[J].J Bacteriol,2007,189(2):305-312.

[26]BACHTIAR B M,COLOE P J,FRY B N.Knockout mutagenesis of the kpsE gene of Campylobacter jejuni 81116 and its involvement in bacterium-host interactions
[J].FEMS Immunol Med Microbiol,2007,49(1):149-154.
[1] WANG Jiajing,GU Haiying. Research progress on genotyping of Helicobacter pylori[J]. Journal of ZheJiang University(Medical Science), 2018, 47(1): 97-103.
[2] JIANG Yiqian, GUO Qingmin, GU Jianzhong, XU Xiaoping, AN Suhong, SU Fang, BAO Yanhong, HUANG Changxin, GUAN Xiaoxiang. Effect of microRNA-29b on proliferation and migration of breast cancer cells and its molecular mechanism[J]. Journal of ZheJiang University(Medical Science), 2017, 46(4): 349-356.
[3] LI Yu, WANG Yueyue, WANG Haifeng, ZHANG Lingyu, DING Yongxing, CHEN Sulian, YANG Qingling, CHEN Changjie. Effects of lncRNA RP11-770J1.3 and TMEM25 expression on paclitaxel resistance in human breast cancer cells[J]. Journal of ZheJiang University(Medical Science), 2017, 46(4): 364-370.
[4] CHEN Dahua, LI Youming. Construction of all-in-one CRISPR/Cas9 vector system targeting miR-101a gene in mouse hepatic cell line AML12[J]. Journal of ZheJiang University(Medical Science), 2017, 46(4): 427-432.
[5] WU Zhihua, JING Min, LIANG Hanying, YANG Rong, HUANG Yaping, CHEN Xiaoming, HU Jianhua, FAN Jun. T cell receptor β-chain CDR3 spectratyping and cytomegalovirus activation in allogeneic hematopoietic stem cell transplant recipients[J]. Journal of ZheJiang University(Medical Science), 2016, 45(5): 515-521.
[6] CHEN Xiaojing, XU Junfen, YE Jing, CHENG Xiaodong, XIE Xing, LYU Weiguo. Expression of miR-let-7e-3p in cervical intraepithelial neoplasm and cervix carcinoma and its clinical significance[J]. Journal of ZheJiang University(Medical Science), 2016, 45(4): 342-348.
[7] ZHOU Qihui, ZHU Biao. Research advances in evaluation and measurement techniques of latent human immunodeficiency virus reservoirs[J]. Journal of ZheJiang University(Medical Science), 2016, 45(3): 256-260.
[8] XIE Hua, HAO Ying, YIN Qiang, LI Wen-bing, LU Hui, JIA Zheng-ping, WANG Rong. Expression of plateau adaptation gene of rat tissues after plain acute exposure to high altitude[J]. Journal of ZheJiang University(Medical Science), 2015, 44(5): 571-577.
[9] LU Xiao-hui,et al. Construction and identification of anti-HER2 phage display single chain fragment of variable region library in human breast cancer[J]. Journal of ZheJiang University(Medical Science), 2014, 43(4): 434-440.
[10] WANG Dan,et al. Relationship between IL-18 gene polymorphism and unexplained recurrent spontaneous abortion [J]. Journal of ZheJiang University(Medical Science), 2014, 43(4): 448-452.
[11] CHAI Xiao-juan, HU Hai-hong, YU Lu-shan, ZENG Su . Expression of human glutathione S-transferase A1, P1 and T1 in Escherichia coli[J]. Journal of ZheJiang University(Medical Science), 2014, 43(2): 168-174.
[12] HE Ping, TAN Li, HU Wei-Wei, DAI Hai-Bin, HU Yong-Zhou, CHEN Zhong. Identification of a HEK-293 cell line containing stably-transfected H3R gene and screening for novel non-imidazole histamine H3 receptor antagonists[J]. Journal of ZheJiang University(Medical Science), 2013, 42(3): 276-282.
[13] WU Yi-Fei, SUN Ai-Hua, ZHAO Jin-Fang, GE Yu-Mei, YAN Jie. Distribution of drug inactive enzyme genes in bacterial isolates and mechanism of its induction and inhibition[J]. Journal of ZheJiang University(Medical Science), 2013, 42(2): 131-140.
[14] ZHENG Lin-Li, GE Yu-Mei, HU Wei-Lin, YAN Jie. Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism[J]. Journal of ZheJiang University(Medical Science), 2013, 42(2): 156-163.
[15] . Expression of RRM1 and ERCC1 genes in tumor tissues and peripheral blood lymphocytes of advanced non-small cell lung cancer[J]. Journal of ZheJiang University(Medical Science), 2012, 41(5): 540-546.