Abstract:The purpose of this study was to construct espF deletion mutant of EHEC O157∶H7 and investigate its properties.espF deletion fragment(△espF)was amplified by two pairs of primers designed according to the sequence of espF and its upper and down streams.The △espF fragment was cloned into suicide plasmid pCVD442 and reconstructed suicide plasmid pCVD442-△espF by conjugating into EHEC O157∶H7 from E.coli SM10λpir.espF deletion mutant was screened with resistance sign,sucrose and PCR.Then the adhesion activity of mutant was compared with the wide type.The sequencing result showed that 162bp of espF was deleted.The adhesion rate of the wide type to LoVo cells was higher than that to the mutant(P<0.001).It's concluded that espF deletion mutant of EHEC O157∶H7 was constructed successfully,terming as EHEC O157∶H7(△espF).The adhesion activity of the mutant was attenuated.espF could promotes EHEC's adhesion to LoVo cells.
[1]Hodges K,Alto NM,Ramaswamy K,et al.The enteropatho-genicEscherichiacolieffector protein EspF decreases sodiumhy-drogen exchanger3activity[J].Cell Microbiol,2008,10(8):1735-45.
[2]Alto NM,Weften AW,Rardin MJ,et al.The type III effector EspF coordinates membrane traficking by the spatiotemporal ac-tivation of two eukaryotic signaling pathways[J].J Cell Biol,2007,178(7):1265-1278.
[3]Dean P,Maresca M,Schüller S,et al.Potent diarrheagenic mechanism mediated by the cooperative action of three entero-pathogenicEscherichia coli-injected effector proteins[J].Proc Natl Acad Sci US A,2006,103(6):1876-81.
[4]Shaw RK,Cleary J,Murphy MS,et al.Interaction of entero-pathogenicEscherichia coliwith human intestinal mucosa:role of effector proteins in brush border remodeling and formation of attaching and effacing lesions[J].Infect Immun,2005,73(2):1243-51.
[5]Nagai T,Abe A,Sasakawa C.Targeting of enteropathogenic EscherichiacoliEspFto host mitochondria is essential for bacte-rial pathogenesis[J].J Biol Chem,2005,280(4):2998-3011.
[6]Viswanathan VK,Koutsouris A,Lukic S,et al.Comparative a-nalysis of EspF from enteropathogenic and enterohemorrhagic Escherichia coliin alteration of epithelial barrier function[J].In-fect Immun,2004,72:3218-3227.
[7]Nougayrede JP,Donnenberg MS.EnteropathogenicEscherichia coliEspFis targeted to mitochondria and is required to initiate the mitochondrial death pathway[J].Cell Microbiol,2004,6:1097-1111
[8]McNamara BP,Koutsouris A,O'Connell CB,et al.Translocated EspF protein from enteropathogenicEscherichia colidisrupts host intestinal barrier function[J].Clin Invest,2001,107(5):621-9
[9]Crane JK,McNamara BP,Donnenberg MS.Role of EspF in host cell deathinduced by enteropathogenicEscherichiacoli[J].Cell Microbiol,2001,3(4):197-211.
[10]Torres AG,Kaper JB.Multiple elements controlling adherence of enterohemorrhagicEscherichiacoliO157∶H7to HeLa cells[J].Infect Immun,2003,71(9):4985-95.
[11]Vlisidou I,Dziva F,Best A,et al.Role of intimin-Tir interac-tions and the Tir-Cytoskeleton coupling protein in the coloniza-tion of calves and lambs byEscherichiacoliO157∶H7[J].In-fect Immun,2006,74:758-764.