单核细胞增多性李斯特菌RsbR抗酸应激机制（英文）
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摘要
目的:探明单增李斯特菌RsbR的两个保守磷酸化位点是否影响SigB(sigma B)的表达。创新点:解析了RsbR在单增李斯特菌中SigB网络调控中的作用。方法:通过基因缺失、定点突变和回补技术构建rsb R基因缺失株、回补株和回补突变菌株,进行酸应激条件下的生长能力和强酸条件下的存活能力试验,并检测在酸应激下菌株中的SigB基因和蛋白表达的改变情况。结论:Rsb R缺失能够显著降低单增李斯特菌的抗酸应激能力和下调SigB表达,Rsb R-175位点是SigB调控途径中Rsb T释放的先决条件。
Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. Rsb R, an upstream regulator of the sigma B(Sig B) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion(?rsb R), complementation(C-?rsb R), and phosphorylation site mutations in the rsbR(RsbR-T175 A, RsbR-T209 A, and RsbR-T175 A-T209 A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsb R reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175 A disabled RsbR complementation, while RsbR-T209 A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of Rsb R are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.
Listeria monocytogenes is an important zoonotic foodborne pathogen that can tolerate a number of environmental stresses. Rsb R, an upstream regulator of the sigma B(Sig B) factor, is thought to sense environmental challenges and trigger the SigB pathway. In Bacillus subtilis, two phosphorylation sites in RsbR are involved in activating the SigB pathway and a feedback mechanism, respectively. In this study, the role of RsbR in L. monocytogenes under mild and severe stresses was investigated. Strains with genetic deletion(?rsb R), complementation(C-?rsb R), and phosphorylation site mutations in the rsbR(RsbR-T175 A, RsbR-T209 A, and RsbR-T175 A-T209 A) were constructed to evaluate the roles of these RsbR sequences in listerial growth and survival. SigB was examined at the transcriptional and translational levels. Deletion of rsb R reduced listerial growxth and survival in response to acidic stress. Substitution of the phosphorylation residue RsbR-T175 A disabled RsbR complementation, while RsbR-T209 A significantly upregulated SigB expression and listerial survival. Our results provide clear evidence that two phosphorylation sites of Rsb R are functional in L. monocytogenes under acidic conditions, similar to the situation in B. subtilis.
引文
Chaturongakul S,Boor KJ,2004.Rsbt and RsbV contribute toσB-dependent survival under environmental,energy,and intracellular stress conditions in Listeria monocytogenes.Appl Environ Microbiol,70(9):5349-5356.https://doi.org/10.1128/AEM.70.9.5349-5356.2004
Chen CC,Yudkin MD,Delumeau O,2004.Phosphorylation and Rsb X-dependent dephosphorylation of RsbR in the Rsb R-Rs Bs complex of Bacillus subtilis.J Bacteriol,186(20):6830-6836.https://doi.org/10.1128/JB.186.20.6830-6836.2004
Chen JS,Cheng CY,Xia Y,et al.,2011.Lmo0036,an ornithine and putrescine carbamoyltransferase in Listeria monocytogenes,participates in arginine deiminase and agmatine deiminase pathways and mediates acid tolerance.Microbiology,157(11):3150-3161.https://doi.org/10.1099/mic.0.049619-0
Cotter PD,Gahan CGM,Hill C,2000.Analysis of the role of the Listeria monocytogenes F0F1-ATPase operon in the acid tolerance response.Int J Food Microbiol,60(2-3):137-146.https://doi.org/10.1016/S0168-1605(00)00305-6
Delumeau O,Chen CC,Murray JW,et al.,2006.Highmolecular-weight complexes of RsbR and paralogues in the environmental signaling pathway of Bacillus subtilis.J Bacteriol,188(22):7885-7892.https://doi.org/10.1128/JB.00892-06
Eymann C,Schulz S,Gronau K,et al.,2011.In vivo phosphorylation patterns of key stressosome proteins define a second feedback loop that limits activation of Bacillus subtilisσB.Mol Microbiol,80(3):798-810.https://doi.org/10.1111/j.1365-2958.2011.07609.x
Gaidenko TA,Bie XM,Baldwin EP,et al.,2012.Two surfaces of a conserved interdomain linker differentially affect output from the RST sensing module of the Bacillus subtilis stressosome.J Bacteriol,194(15):3913-3921.https://doi.org/10.1128/JB.00583-12
Hecker M,Pané-FarréJ,V?lker U,2007.Sigb-dependent general stress response in bacillus subtilis and related Gram-positive bacteria.Annu Rev Microbiol,61:215-236.https://doi.org/10.1146/annurev.micro.61.080706.093445
Hecker M,Reder A,Fuchs S,et al.,2009.Physiological proteomics and stress/starvation responses in Bacillus subtilis and staphylococcus aureus.Res Microbiol,160(4):245-258.https://doi.org/10.1016/j.resmic.2009.03.008
Kim TJ,Gaidenko TA,Price CW,2004a.A multicomponent protein complex mediates environmental stress signaling in Bacillus subtilis.J Mol Biol,341(1):135-150.https://doi.org/10.1016/j.jmb.2004.05.043
Kim TJ,Gaidenko TA,Price CW,2004b.In vivo phosphorylation of partner switching regulators correlates with stress transmission in the environmental signaling pathway of Bacillus subtilis.J Bacteriol,186(18):6124-6132.https://doi.org/10.1128/JB.186.18.6124-6132.2004
Liebal UW,Thomas M,Jon MW,et al.,2013.Simulations of stressosome activation emphasize allosteric interactions between RsbR and RsbT.BMC Syst Biol,7:3.https://doi.org/10.1186/1752-0509-7-3
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT.Methods,25(4):402-408.https://doi.org/10.1006/meth.2001.1262
Marles-Wright J,Grant T,Delumeau O,et al.,2008.Molecular architecture of the“stressosome,”a signal integration and transduction hub.Science,322(5898):92-96.https://doi.org/10.1126/science.1159572
Martinez L,Reeves A,Haldenwang W,2010.Stressosomes formed in Bacillus subtilis from the RsbR protein of Listeria monocytogenes allowσB activation following exposure to either physical or nutritional stress.J Bacteriol,192(23):6279-6286.https://doi.org/10.1128/JB.00467-10
Nic Aogáin K,O'Byrne CP,2016.The role of stress and stress adaptations in determining the fate of the bacterial pathogen Listeria monocytogenes in the food chain.Front Microbiol,7:1865.https://doi.org/10.3389/fmicb.2016.01865
O'Byrne CP,Karatzas KAG,2008.Chapter 5:the role of sigma B(σB)in the stress adaptations of Listeria monocytogenes:overlaps between stress adaptation and virulence.Adv Appl Microbiol,65:115-140.https://doi.org/10.1016/S0065-2164(08)00605-9
Okpo E,Leith J,Smith-Palmer A,et al.,2015.An outbreak of an unusual strain of Listeria monocytogenes infection in North-East Scotland.J Infect Public Health,8(6):612-618.https://doi.org/10.1016/j.jiph.2015.05.009
Pané-FarréJ,Lewis RJ,Stülke J,2005.The Rsb RST stress module in bacteria:a signalling system that may interact with different output modules.J Mol Microbiol Biotechnol,9:65-76.https://doi.org/10.1159/000088837
Price CW,2011.General stress response in Bacillus subtilis and related Gram-positive bacteria.In:Storz G,Hengge R(Eds.),Bacterial Stress Responses,2nd Ed.American Society of Microbiology(ASM)Press,Washington,DC,p.301-318.https://doi.org/10.1128/9781555816841.ch17
Shan Y,Liu ZQ,Li GW,et al.,2018.Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λproduction by blocking the nuclear factor-κB nuclear translocation.J Zhejiang Univ-Sci B(Biomed&Biotechnol),19(8):570-580.https://doi.org/10.1631/jzus.B1700283
Shin JH,Brody MS,Price CW,2010.Physical and antibiotic stresses require activation of the RsbU phosphatase to induce the general stress response in Listeria monocytogenes.Microbiology,156:2660-2669.https://doi.org/10.1099/mic.0.041202-0
Varma JK,Samuel MC,Marcus R,et al.,2007.Listeria monocytogenes infection from foods prepared in a commercial establishment:a case-control study of potential sources of sporadic illness in the united states.Clin Inf Dis,44(4):521-528.https://doi.org/10.1086/509920
Xia Y,Xin YP,Li XL,et al.,2016.To modulate survival under secondary stress conditions,Listeria monocytogenes10403S employs RsbX to downregulateσB activity in the poststress recovery stage or stationary phase.Appl Environ Microbiol,82(4):1126-1135.https://doi.org/10.1128/AEM.03218-15
Zhang ZC,Meng QL,Qiao J,et al.,2013.RsbV of Listeria monocytogenes contributes to regulation of environmental stress and virulence.Arch Microbiol,195(2):113-120.https://doi.org/10.1007/s00203-012-0855-5
Zhou YS,Gu YX,Qi BZ,et al.,2017.Porcine circovirus type 2capsid protein induces unfolded protein response with subsequent activation of apoptosis.J Zhejiang Univ-Sci B(Biomed&Biotechnol),18(4):316-323.https://doi.org/10.1631/jzus.B1600208
Chen CC,Yudkin MD,Delumeau O,2004.Phosphorylation and Rsb X-dependent dephosphorylation of RsbR in the Rsb R-Rs Bs complex of Bacillus subtilis.J Bacteriol,186(20):6830-6836.https://doi.org/10.1128/JB.186.20.6830-6836.2004
Chen JS,Cheng CY,Xia Y,et al.,2011.Lmo0036,an ornithine and putrescine carbamoyltransferase in Listeria monocytogenes,participates in arginine deiminase and agmatine deiminase pathways and mediates acid tolerance.Microbiology,157(11):3150-3161.https://doi.org/10.1099/mic.0.049619-0
Cotter PD,Gahan CGM,Hill C,2000.Analysis of the role of the Listeria monocytogenes F0F1-ATPase operon in the acid tolerance response.Int J Food Microbiol,60(2-3):137-146.https://doi.org/10.1016/S0168-1605(00)00305-6
Delumeau O,Chen CC,Murray JW,et al.,2006.Highmolecular-weight complexes of RsbR and paralogues in the environmental signaling pathway of Bacillus subtilis.J Bacteriol,188(22):7885-7892.https://doi.org/10.1128/JB.00892-06
Eymann C,Schulz S,Gronau K,et al.,2011.In vivo phosphorylation patterns of key stressosome proteins define a second feedback loop that limits activation of Bacillus subtilisσB.Mol Microbiol,80(3):798-810.https://doi.org/10.1111/j.1365-2958.2011.07609.x
Gaidenko TA,Bie XM,Baldwin EP,et al.,2012.Two surfaces of a conserved interdomain linker differentially affect output from the RST sensing module of the Bacillus subtilis stressosome.J Bacteriol,194(15):3913-3921.https://doi.org/10.1128/JB.00583-12
Hecker M,Pané-FarréJ,V?lker U,2007.Sigb-dependent general stress response in bacillus subtilis and related Gram-positive bacteria.Annu Rev Microbiol,61:215-236.https://doi.org/10.1146/annurev.micro.61.080706.093445
Hecker M,Reder A,Fuchs S,et al.,2009.Physiological proteomics and stress/starvation responses in Bacillus subtilis and staphylococcus aureus.Res Microbiol,160(4):245-258.https://doi.org/10.1016/j.resmic.2009.03.008
Kim TJ,Gaidenko TA,Price CW,2004a.A multicomponent protein complex mediates environmental stress signaling in Bacillus subtilis.J Mol Biol,341(1):135-150.https://doi.org/10.1016/j.jmb.2004.05.043
Kim TJ,Gaidenko TA,Price CW,2004b.In vivo phosphorylation of partner switching regulators correlates with stress transmission in the environmental signaling pathway of Bacillus subtilis.J Bacteriol,186(18):6124-6132.https://doi.org/10.1128/JB.186.18.6124-6132.2004
Liebal UW,Thomas M,Jon MW,et al.,2013.Simulations of stressosome activation emphasize allosteric interactions between RsbR and RsbT.BMC Syst Biol,7:3.https://doi.org/10.1186/1752-0509-7-3
Livak KJ,Schmittgen TD,2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT.Methods,25(4):402-408.https://doi.org/10.1006/meth.2001.1262
Marles-Wright J,Grant T,Delumeau O,et al.,2008.Molecular architecture of the“stressosome,”a signal integration and transduction hub.Science,322(5898):92-96.https://doi.org/10.1126/science.1159572
Martinez L,Reeves A,Haldenwang W,2010.Stressosomes formed in Bacillus subtilis from the RsbR protein of Listeria monocytogenes allowσB activation following exposure to either physical or nutritional stress.J Bacteriol,192(23):6279-6286.https://doi.org/10.1128/JB.00467-10
Nic Aogáin K,O'Byrne CP,2016.The role of stress and stress adaptations in determining the fate of the bacterial pathogen Listeria monocytogenes in the food chain.Front Microbiol,7:1865.https://doi.org/10.3389/fmicb.2016.01865
O'Byrne CP,Karatzas KAG,2008.Chapter 5:the role of sigma B(σB)in the stress adaptations of Listeria monocytogenes:overlaps between stress adaptation and virulence.Adv Appl Microbiol,65:115-140.https://doi.org/10.1016/S0065-2164(08)00605-9
Okpo E,Leith J,Smith-Palmer A,et al.,2015.An outbreak of an unusual strain of Listeria monocytogenes infection in North-East Scotland.J Infect Public Health,8(6):612-618.https://doi.org/10.1016/j.jiph.2015.05.009
Pané-FarréJ,Lewis RJ,Stülke J,2005.The Rsb RST stress module in bacteria:a signalling system that may interact with different output modules.J Mol Microbiol Biotechnol,9:65-76.https://doi.org/10.1159/000088837
Price CW,2011.General stress response in Bacillus subtilis and related Gram-positive bacteria.In:Storz G,Hengge R(Eds.),Bacterial Stress Responses,2nd Ed.American Society of Microbiology(ASM)Press,Washington,DC,p.301-318.https://doi.org/10.1128/9781555816841.ch17
Shan Y,Liu ZQ,Li GW,et al.,2018.Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λproduction by blocking the nuclear factor-κB nuclear translocation.J Zhejiang Univ-Sci B(Biomed&Biotechnol),19(8):570-580.https://doi.org/10.1631/jzus.B1700283
Shin JH,Brody MS,Price CW,2010.Physical and antibiotic stresses require activation of the RsbU phosphatase to induce the general stress response in Listeria monocytogenes.Microbiology,156:2660-2669.https://doi.org/10.1099/mic.0.041202-0
Varma JK,Samuel MC,Marcus R,et al.,2007.Listeria monocytogenes infection from foods prepared in a commercial establishment:a case-control study of potential sources of sporadic illness in the united states.Clin Inf Dis,44(4):521-528.https://doi.org/10.1086/509920
Xia Y,Xin YP,Li XL,et al.,2016.To modulate survival under secondary stress conditions,Listeria monocytogenes10403S employs RsbX to downregulateσB activity in the poststress recovery stage or stationary phase.Appl Environ Microbiol,82(4):1126-1135.https://doi.org/10.1128/AEM.03218-15
Zhang ZC,Meng QL,Qiao J,et al.,2013.RsbV of Listeria monocytogenes contributes to regulation of environmental stress and virulence.Arch Microbiol,195(2):113-120.https://doi.org/10.1007/s00203-012-0855-5
Zhou YS,Gu YX,Qi BZ,et al.,2017.Porcine circovirus type 2capsid protein induces unfolded protein response with subsequent activation of apoptosis.J Zhejiang Univ-Sci B(Biomed&Biotechnol),18(4):316-323.https://doi.org/10.1631/jzus.B1600208