OmpR对伤寒沙门菌侵袭上皮细胞能力的影响
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摘要
目的:研究OmpR对伤寒沙门菌侵袭力的影响及相关分子机制。方法:利用空载体对照株(WT-pBAD33)、ompR缺陷株(ΔompR-pBAD33)和ompR回补株(C-ΔompR)进行HeLa细胞侵袭实验,探究伤寒沙门菌OmpR对于HeLa细胞侵袭力的影响。进一步用荧光实时定量PCR(qRT-PCR)、凝胶阻滞实验(EMSA)分析伤寒沙门菌OmpR对侵袭相关基因iagA、invF、prgH的调控作用。结果:成功制备伤寒沙门菌C-ΔompR。在HeLa细胞侵袭实验中,ΔompR-pBAD33侵袭力显著低于WT-pBAD33(P <0.01),而C-ΔompR侵袭能力较ΔompR-pBAD33明显升高(P <0.01); qRTPCR结果显示,ΔompR-pBAD33中侵袭相关基因iagA、invF、prgH的转录水平较WT-pBAD33明显下降(P <0.01); EMSA结果表明,OmpR可直接与prgH基因启动子区域结合,与iagA和invF基因启动子区域未有结合。结论:OmpR通过直接调节prgH和间接调节iagA、invF增加侵袭相关基因的表达,从而增强伤寒沙门菌对上皮细胞的侵袭力。
Objective: To investigate the effect of OmpR on the invasion of Salmonella enterica serovar Typhi(S. Typhi) and its potential mechanism. Methods: The invasion of S. Typhi were investigated by using cell invasion experiment with the empty vector strain(WT-pBAD33),the ompR deletion mutant strain(ΔompR-pBAD33) and the ompR complementary strain(C-ΔompR). In addition,qRT-PCR and EMSA were used to analyze the regulation of OmpR on the expression of invasion-related genes iagA,inv F and prgH. Results: In the invasion assay,ΔompR-pBAD33 showed significantly weaker invasion than that of WT-pBAD33(P < 0.01),and the invasion of C-ΔompR was higher than that of ΔompR-pBAD33(P < 0.01); expressional levels of invasion-related genes in ΔompR-pBAD33 were lower than those in WT-pBAD33(P < 0.01). EMSA results showed that OmpR bound to the promoter region of prgH in a dose-dependent manner,but it was unable to bind to the upstream regions of iagA and invF. Conclusion: OmpR promotes the invasion of S. Typhi,which results from up-regulating the expression of invasion-related genes by directly regulating prgH,indirectly regulating iagA and invF.
Objective: To investigate the effect of OmpR on the invasion of Salmonella enterica serovar Typhi(S. Typhi) and its potential mechanism. Methods: The invasion of S. Typhi were investigated by using cell invasion experiment with the empty vector strain(WT-pBAD33),the ompR deletion mutant strain(ΔompR-pBAD33) and the ompR complementary strain(C-ΔompR). In addition,qRT-PCR and EMSA were used to analyze the regulation of OmpR on the expression of invasion-related genes iagA,inv F and prgH. Results: In the invasion assay,ΔompR-pBAD33 showed significantly weaker invasion than that of WT-pBAD33(P < 0.01),and the invasion of C-ΔompR was higher than that of ΔompR-pBAD33(P < 0.01); expressional levels of invasion-related genes in ΔompR-pBAD33 were lower than those in WT-pBAD33(P < 0.01). EMSA results showed that OmpR bound to the promoter region of prgH in a dose-dependent manner,but it was unable to bind to the upstream regions of iagA and invF. Conclusion: OmpR promotes the invasion of S. Typhi,which results from up-regulating the expression of invasion-related genes by directly regulating prgH,indirectly regulating iagA and invF.
引文
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[11]Ellermeier CD,Ellermeier JR,Slauch JM.Hil D,Hil Cand Rts A constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hil A in Salmonella enterica serovar Typhimurium[J].Mol Microbiol,2005,57(3):691-705.
[12]Lara-Tejero M,Galn JE.Salmonella enterica Serovar typhimurium pathogenicity island 1-encoded typeⅢsecretion system translocases mediate intimate attachment to nonphagocytic cells[J].Infect Immun,2009,77(7):2635-2642.
[13]Bergeron JRC,Brockerman JA,Vuckovic M,et al.Characterization of the two conformations adopted by the T3SS inner-membrane protein Prg K[J].Protein Sci,2018,27(9):1680-1691.
[2]Oropeza R,Calva E.The cysteine 354 and 277 residues of Salmonella enterica serovar Typhi Env Z are determinants of autophosphorylation and OmpR phosphorylation[J].FEMS Microbiol Lett,2009,292(2):282-290.
[3]F1brega A,Vila J.Salmonella enterica serovar Typhimurium skills to succeed in the host:virulence and regulation[J].Clin Microbiol Rev,2013,26(2):308-341.
[4]Virlogeux-Payant I,Baucheron S,Pelet J,et al.Tol C,but not Acr B,is involved in the invasiveness of multidrug-resistant Salmonella enterica serovar Typhimurium by increasing typeⅢsecretion system-1 expression[J].Int J Med Microbiol,2008,298(7/8):561-569.
[5]Bustamante VH,Martínez LC,Santana FJ,et al.Hil D-mediated transcriptional cross-talk between SPI-1 and SPI-2[J].Proc Natl Acad Sci,2008,105(38):14591-14596.
[6]黄新祥,徐顺高,周丽萍,等.伤寒沙门菌OmpR缺陷变异株制备与分泌蛋白初析[J].世界感染杂志,2005,5(3):185-188.
[7]Parry CM,Hien TT,Dougan G,et al.Typhoid fever[J].N Engl J Med,2002,347(22):1770-1782.
[8]Livermore TM,Chubb JR,Saiardi A.Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum[J].Proc Natl Acad Sci U S A,2016,113(4):996-1001.
[9]Zhang Y,Xia L,Lin L,et al.Reciprocal regulation of OmpR and Hfq and their regulatory actions on the Vi polysaccharide capsular antigen in Salmonella enterica Serovar Typhi[J].Curr Microbiol,2018,75(6):773-778.
[10]Bajaj V,Hwang C,Lee CA.hil A is a novel ompR/toxRfamily member that activates the expression of Salmonella typhimurium invasion genes[J].Mol Microbiol,1995,18(4):715-727.
[11]Ellermeier CD,Ellermeier JR,Slauch JM.Hil D,Hil Cand Rts A constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hil A in Salmonella enterica serovar Typhimurium[J].Mol Microbiol,2005,57(3):691-705.
[12]Lara-Tejero M,Galn JE.Salmonella enterica Serovar typhimurium pathogenicity island 1-encoded typeⅢsecretion system translocases mediate intimate attachment to nonphagocytic cells[J].Infect Immun,2009,77(7):2635-2642.
[13]Bergeron JRC,Brockerman JA,Vuckovic M,et al.Characterization of the two conformations adopted by the T3SS inner-membrane protein Prg K[J].Protein Sci,2018,27(9):1680-1691.
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