鼠伤寒沙门氏菌群体感应复苏oxyR基因缺失引起的VBNC状态
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摘要
探讨鼠伤寒沙门氏菌oxyR基因缺失株引起的VBNC状态及其与群体感应的关系。运用同源重组的方法构建oxyR基因无痕缺失的鼠伤寒沙门氏菌并检测该菌株对H_2O_2的敏感性;将oxyR基因缺失株和亲本株(WT)涂布或滴于LB固体培养基,观察其是否生长及其浓度依赖性生长情况;用swimming和swarming平板检测oxyR基因缺失株和WT的运动能力;检测固体培养基和液体培养基中沙门氏菌分解H_2O_2的能力。成功构建了oxyR无痕缺失菌株;oxyR基因缺失株在0.1 mmol/L H_2O_2的LB平板上形成的菌苔发生了变形,在1 mmol/L H_2O_2的LB平板上不能生长,而WT均能生长;6×10~6和6×10~5 cfu/mL的WT涂布于LB平板上能长满菌苔,而等量的oxyR缺失株不能生长菌落;不同浓度的WT滴于LB平板均能形成菌苔,而oxyR缺失株仅在OD_(600)≥10~(-1)浓度时才能形成菌苔;oxyR缺失株泳动距离无显著性变化,而群集运动距离显著性大于WT;固体培养的沙门氏菌比液体培养的沙门氏菌有更强的分解H_2O_2的能力。鼠伤寒沙门氏菌的群体感应系统通过调控其群集运动和H_2O_2分解能力来复苏由oxyR基因缺失引起的VBNC状态。
VBNC status caused by oxyR deletion and the relationship with group sensing in Salmonella typhimurium was investigated. Homogenetic combination method was used to construct oxyR gene traceless deletion S. typhimurium strain and check its hydrogen peroxide sensibility. The oxyR deletion strain and parent spread or dropped on LB solid media was observed if their growth depend on their concentration. Swimming and swarming plate were used to check moving ability of oxyR deletion strain and parent strain, and determined the ability of Salmonella to decompose H_2O_2 solid and liquid media. The results oxyR traceless deletion strain was successfully constructed. The bacterial lawn of oxyR deletion strain formed on LB plate with 0.1 mmol/L H_2O_2 was distorted, and cannot grow on LB plate with 1 mmol/L H_2O_2, but the parent strain can grow,while the WT had growth under the same condition. 6×10~6 or 6×10~5 cfu/mL oxyR mutant strain were inoculated onto LB plates and could not grow. Only if its concentration great than or equal to OD_(600) 0.1, the oxyR deletion strain could form bacterial lawn on LB plates. For the parent strain, it can grow in both cases. The swimming distance of the oxyR deletion strain had no significant change while its swarming distance increased obviously. The H_2O_2 decomposing ability of the strain collected from plate has significant increase than that of the strain collected from broth. Group sensing systems up-regulate the swarming ability and H_2O_2 decomposing ability to resuscitate VBNC caused by oxyR deletion.
VBNC status caused by oxyR deletion and the relationship with group sensing in Salmonella typhimurium was investigated. Homogenetic combination method was used to construct oxyR gene traceless deletion S. typhimurium strain and check its hydrogen peroxide sensibility. The oxyR deletion strain and parent spread or dropped on LB solid media was observed if their growth depend on their concentration. Swimming and swarming plate were used to check moving ability of oxyR deletion strain and parent strain, and determined the ability of Salmonella to decompose H_2O_2 solid and liquid media. The results oxyR traceless deletion strain was successfully constructed. The bacterial lawn of oxyR deletion strain formed on LB plate with 0.1 mmol/L H_2O_2 was distorted, and cannot grow on LB plate with 1 mmol/L H_2O_2, but the parent strain can grow,while the WT had growth under the same condition. 6×10~6 or 6×10~5 cfu/mL oxyR mutant strain were inoculated onto LB plates and could not grow. Only if its concentration great than or equal to OD_(600) 0.1, the oxyR deletion strain could form bacterial lawn on LB plates. For the parent strain, it can grow in both cases. The swimming distance of the oxyR deletion strain had no significant change while its swarming distance increased obviously. The H_2O_2 decomposing ability of the strain collected from plate has significant increase than that of the strain collected from broth. Group sensing systems up-regulate the swarming ability and H_2O_2 decomposing ability to resuscitate VBNC caused by oxyR deletion.
引文
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[11] Zheng M, Aslund F, Storz G. Activation of the OxyR transcription factor by reversible disulfide bond formation[J]. Science, 1998, 279: 1718-1721.
[12] Lee C, Lee SM, Mukhopadhyay P, et al. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path[J]. Nature Structural & Molecular Biology, 2004, 11: 1179-1185.
[13] Oliver JD. Recent findings on the viable but nonculturable state in pathogenic bacteria[J]. Fems Microbiology Reviews, 2010, 34: 415-425.
[14] Oliver JD. The viable but nonculturable state in bacteria[J]. The journal of Microbiology, 2005, 43: 93-100.
[15] Whitesides MD, Oliver JD. Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State[J]. Applied and Environmental Microbiology, 1997, 63: 1002-1005.
[16] Liu Y, Wang C, Tyrrell G, et al. Induction of Escherichia coli O157:H7 into the viable but non-culturable state by chloraminated water and river water, and subsequent resuscitation[J]. Environmental Microbiology Reports, 2009, 1: 155-161.
[17] Zhao L, Matthews KR. Influence of starvation, temperature, and pH on culturability of Escherichia coli O157:H7[J]. Journal of Food Safety, 2000, 20: 193-206.
[18] Signoretto C, Burlacchini G, Lleo MM, et al. Adhesion of Enterococcus faecalis in the nonculturable state to plankton is the main mechanism responsible for persistence of this bacterium in both lake and seawater[J]. Applied and Environmental Microbiology, 2004, 70: 6892-6896.
[19] Morishige Y, Fujimori K, Amano F. Differential resuscitative effect of pyruvate and its analogues on VBNC (viable but non-culturable) Salmonella[J]. Microbes and Environments, 2013, 28: 180-186.
[20] Senoh M, Ghosh-Banerjee J, Ramamurthy T, et al. Conversion of viable but nonculturable Vibrio cholerae to the culturable state by co-culture with eukaryotic cells[J]. Microbiology and Immunology, 2010, 54: 502-507.
[21] Ayrapetyan M, Williams TC, Oliver JD. Interspecific quorum sensing mediates the resuscitation of viable but nonculturable vibrios[J]. Applied and Environmental Microbiology, 2014, 80: 2478-2483.
[22] Kendall MM, Sperandio V. Cell-to-Cell Signaling in Escherichia coli and Salmonella[J]. EcoSal Plus, 2014, 6(1): 10.
[23] Jesudhasan PR, Cepeda ML, Widmer K, et al. Transcriptome analysis of genes controlled by luxS/autoinducer-2 in Salmonella enterica serovar Typhimurium[J]. Foodborne Pathogens and Disease, 2010, 7: 399-410.
[24] Burbank L, Roper MC. OxyR and SoxR modulate the inducible oxidative stress response and are implicated during different stages of infection for the bacterial phytopathogen Pantoea stewartii subsp. stewartii[J]. Molecular Plant-Microbe Interactions, 2014, 27: 479-490.
[25] Rocha ER, Owens G, Jr., Smith CJ. The redox-sensitive transcriptional activator OxyR regulates the peroxide response regulon in the obligate anaerobe Bacteroides fragilis[J]. Journal of Bacteriology, 2000, 182: 5059-5069.
[26] Kim JA, Mayfield J. Identification of Brucella abortus OxyR and its role in control of catalase expression[J]. Journal of Bacteriology, 2000, 182: 5631-5633.
[27] Xie K, Peng H, Hu H, et al. OxyR, an important oxidative stress regulator to phenazines production and hydrogen peroxide resistance in Pseudomonas chlororaphis VGP72[J]. Microbiological Research, 2013, 168: 646-653.
[28] Christman MF, Morgan RW, Jacobson FS, et al. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium[J]. Cell, 1985, 41: 753-762.
[29] Vattanaviboon P, Whangsuk W, Mongkolsuk S. A suppressor of the menadione-hypersensitive phenotype of a Xanthomonas campestris pv. phaseoli oxyR mutant reveals a novel mechanism of toxicity and the protective role of alkyl hydroperoxide reductase[J]. Journal of Bacteriology, 2003, 185: 1734-1738.
[30] Hassett DJ, Ma JF, Elkins JG, et al. Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide[J]. Molecular Microbiology, 1999, 34: 1082-1093.
[31] O′Grady EP, Viteri DF, Malott RJ, et al. Reciprocal regulation by the CepIR and CciIR quorum sensing systems in Burkholderia cenocepacia[J]. BMC Genomics, 2009, 10: 441.
[2] Liu X, Liu Q, Xiao K, et al. Attenuated Salmonella typhimurium delivery of a novel DNA vaccine induces immune responses and provides protection against duck enteritis virus[J]. Veterinary Microbiology, 2016, 186: 189-198.
[3] Huang C, Liu Q, Luo Y, et al. Regulated delayed synthesis of lipopolysaccharide and enterobacterial common antigen of Salmonella typhimurium enhances immunogenicity and cross-protective efficacy against heterologous Salmonella challenge[J]. Vaccine, 2016, 34: 4285-4292.
[4] Vishwakarma V, Sahoo SS, Das S, et al. Cholera toxin-B (ctxB) antigen expressing Salmonella typhimurium polyvalent vaccine exerts protective immune response against Vibrio cholerae infection[J]. Vaccine, 2015, 33: 1880-1889.
[5] Wang L, Wang X, Bi K, et al. Oral Vaccination with Attenuated Salmonella typhimurium-Delivered TsPmy DNA Vaccine Elicits Protective Immunity against Trichinella spiralis in BALB/c Mice[J]. PLoS Neglected Tropical Diseases, 2016, 10: e0004952.
[6] Ding J, Zheng Y, Wang Y, et al. Immune responses to a recombinant attenuated Salmonella typhimurium strain expressing a Taenia solium oncosphere antigen TSOL18[J]. Comparative Immunology Microbiology and Infectious Diseases, 2013, 36: 17-23.
[7] Nguyen VH, Kim HS, Ha JM, et al. Genetically engineered Salmonella typhimurium as an imageable therapeutic probe for cancer[J]. Cancer Research, 2010, 70: 18-23.
[8] Blache CA, Manuel ER, Kaltcheva TI, et al. Systemic delivery of Salmonella typhimurium transformed with IDO shRNA enhances intratumoral vector colonization and suppresses tumor growth[J]. Cancer Research, 2012, 72: 6447-6456.
[9] Whitby PW, Morton DJ, Vanwagoner TM, et al. Haemophilus influenzae OxyR: characterization of its regulation, regulon and role in fitness[J]. PLoS One, 2012, 7: e50588.
[10] 汪保卫, 施庆珊, 欧阳友生, 等. 细菌抗氧化系统-oxyR 调节子研究进展[J]. 微生物学报, 2008, 48: 1556-1561.
[11] Zheng M, Aslund F, Storz G. Activation of the OxyR transcription factor by reversible disulfide bond formation[J]. Science, 1998, 279: 1718-1721.
[12] Lee C, Lee SM, Mukhopadhyay P, et al. Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path[J]. Nature Structural & Molecular Biology, 2004, 11: 1179-1185.
[13] Oliver JD. Recent findings on the viable but nonculturable state in pathogenic bacteria[J]. Fems Microbiology Reviews, 2010, 34: 415-425.
[14] Oliver JD. The viable but nonculturable state in bacteria[J]. The journal of Microbiology, 2005, 43: 93-100.
[15] Whitesides MD, Oliver JD. Resuscitation of Vibrio vulnificus from the Viable but Nonculturable State[J]. Applied and Environmental Microbiology, 1997, 63: 1002-1005.
[16] Liu Y, Wang C, Tyrrell G, et al. Induction of Escherichia coli O157:H7 into the viable but non-culturable state by chloraminated water and river water, and subsequent resuscitation[J]. Environmental Microbiology Reports, 2009, 1: 155-161.
[17] Zhao L, Matthews KR. Influence of starvation, temperature, and pH on culturability of Escherichia coli O157:H7[J]. Journal of Food Safety, 2000, 20: 193-206.
[18] Signoretto C, Burlacchini G, Lleo MM, et al. Adhesion of Enterococcus faecalis in the nonculturable state to plankton is the main mechanism responsible for persistence of this bacterium in both lake and seawater[J]. Applied and Environmental Microbiology, 2004, 70: 6892-6896.
[19] Morishige Y, Fujimori K, Amano F. Differential resuscitative effect of pyruvate and its analogues on VBNC (viable but non-culturable) Salmonella[J]. Microbes and Environments, 2013, 28: 180-186.
[20] Senoh M, Ghosh-Banerjee J, Ramamurthy T, et al. Conversion of viable but nonculturable Vibrio cholerae to the culturable state by co-culture with eukaryotic cells[J]. Microbiology and Immunology, 2010, 54: 502-507.
[21] Ayrapetyan M, Williams TC, Oliver JD. Interspecific quorum sensing mediates the resuscitation of viable but nonculturable vibrios[J]. Applied and Environmental Microbiology, 2014, 80: 2478-2483.
[22] Kendall MM, Sperandio V. Cell-to-Cell Signaling in Escherichia coli and Salmonella[J]. EcoSal Plus, 2014, 6(1): 10.
[23] Jesudhasan PR, Cepeda ML, Widmer K, et al. Transcriptome analysis of genes controlled by luxS/autoinducer-2 in Salmonella enterica serovar Typhimurium[J]. Foodborne Pathogens and Disease, 2010, 7: 399-410.
[24] Burbank L, Roper MC. OxyR and SoxR modulate the inducible oxidative stress response and are implicated during different stages of infection for the bacterial phytopathogen Pantoea stewartii subsp. stewartii[J]. Molecular Plant-Microbe Interactions, 2014, 27: 479-490.
[25] Rocha ER, Owens G, Jr., Smith CJ. The redox-sensitive transcriptional activator OxyR regulates the peroxide response regulon in the obligate anaerobe Bacteroides fragilis[J]. Journal of Bacteriology, 2000, 182: 5059-5069.
[26] Kim JA, Mayfield J. Identification of Brucella abortus OxyR and its role in control of catalase expression[J]. Journal of Bacteriology, 2000, 182: 5631-5633.
[27] Xie K, Peng H, Hu H, et al. OxyR, an important oxidative stress regulator to phenazines production and hydrogen peroxide resistance in Pseudomonas chlororaphis VGP72[J]. Microbiological Research, 2013, 168: 646-653.
[28] Christman MF, Morgan RW, Jacobson FS, et al. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium[J]. Cell, 1985, 41: 753-762.
[29] Vattanaviboon P, Whangsuk W, Mongkolsuk S. A suppressor of the menadione-hypersensitive phenotype of a Xanthomonas campestris pv. phaseoli oxyR mutant reveals a novel mechanism of toxicity and the protective role of alkyl hydroperoxide reductase[J]. Journal of Bacteriology, 2003, 185: 1734-1738.
[30] Hassett DJ, Ma JF, Elkins JG, et al. Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide[J]. Molecular Microbiology, 1999, 34: 1082-1093.
[31] O′Grady EP, Viteri DF, Malott RJ, et al. Reciprocal regulation by the CepIR and CciIR quorum sensing systems in Burkholderia cenocepacia[J]. BMC Genomics, 2009, 10: 441.