鼠伤寒沙门菌5'-nucleotidase基因缺失株的构建及其生物学特性的研究
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摘要
为了探究5'-nucleotidase基因的功能及其在鼠伤寒沙门菌感染中的作用,本研究首先构建缺失1 112 bp的5'-nucleotidase基因的重组自杀性质粒pR E 112Δ5'-nucleotidase,利用重组自杀性质粒介导的等位基因交换技术,两步法筛选出SL1344Δ5'-nucleotidase缺失株,并对其生物学特性进行初步研究。PCR及测序结果表明,SL1344Δ5'-nucleotidase构建成功,且回复株SL1344CΔ5'-nucleotidase构建成功。进一步研究表明,缺失株SL1344Δ5'-nucleotidase保留了亲本菌株SL1344的血清型1,4,5,12∶i∶1,2,且能够稳定遗传缺失1 112 bp的5'-nucleotidase基因,生长速度没有发生明显改变。但是,缺失株SL1344Δ5'-nucleotidase口服感染6周龄BALB/c小鼠的LD50为4.30×10~7CF U,毒力降低至亲本株SL1344的1.96%。免疫保护率试验结果显示,对6周龄BALB/c小鼠免疫后第17天,鼠伤寒沙门菌野生株攻毒有50%的保护率。缺失株免疫小鼠后第21天,血清抗体IgG水平达到最高。回复菌株与亲本亲株之间生物学特性无显著性差异,基本恢复到野生型水平。上述结果表明,SL1344Δ5'-nucleotidase基因缺失株构建成功,遗传稳定,毒力明显降低,且具有较好的免疫原性,为研究鼠伤寒沙门菌5'-nucleotidase基因与其致病力之间的关系提供了依据,并为研究它在鼠伤寒沙门菌感染中的作用奠定了基础。
To explore the function of the 5'-nucleotidase gene and its role in Salmonella typhimurium infection,the SL1344Δ5'-nucleotidase mutant without 1 112 bp of Salmonella typhimurium recombinant suicide plasmid,and complemented mutant was constructed.Then,the biological characteristics of the mutant were detected. The results of PCR and sequencing showed that the SL1344Δ 5'-nucleotidase and the complemented strain were successfully constructed. The identification results suggested that the serotype of the strain was 1,4,5,12 ∶ i ∶ 1,2,identical to the parent SL1344 strain.In addition,the biochemical characteristics and growth rate did not significantly change and the strain was stable with the recombinant Δ5'-nucleotidase gene in vitro.Compared with the parent SL1344 strain,the virulence of the SL1344Δ5'-nucleotidase was attenuated to BALB/c mice.The mouse lethal test was 4.30×10~7 CFU,showing that the virulence of the strain was 1.98% of SL1344. The immunoprotective efficacy results showed that the protection rate of the animals infected with Salmonella typhimurium was 50% on the immunization by mutant strain.The serum antibodies peaked 21 st d post-immunization. The complemented strain was no significant difference in the biological characteristics with the parent SL1344 strain,and it basically returned to the wild-type level.The SL1344Δ5'-nucleotidase mutant was constructed successfully,and genetic stability,significantly reduced virulence.The above-mentioned results showed that 5'-nucleotidase plays an important role in regulating the virulence of SL1344,which provide a method for studying the relationship between the 5'-nucleotidase gene of Salmonella typhimurium and its pathogenicity,and lay a foundation for studying its role in Salmonella typhimurium infection.
To explore the function of the 5'-nucleotidase gene and its role in Salmonella typhimurium infection,the SL1344Δ5'-nucleotidase mutant without 1 112 bp of Salmonella typhimurium recombinant suicide plasmid,and complemented mutant was constructed.Then,the biological characteristics of the mutant were detected. The results of PCR and sequencing showed that the SL1344Δ 5'-nucleotidase and the complemented strain were successfully constructed. The identification results suggested that the serotype of the strain was 1,4,5,12 ∶ i ∶ 1,2,identical to the parent SL1344 strain.In addition,the biochemical characteristics and growth rate did not significantly change and the strain was stable with the recombinant Δ5'-nucleotidase gene in vitro.Compared with the parent SL1344 strain,the virulence of the SL1344Δ5'-nucleotidase was attenuated to BALB/c mice.The mouse lethal test was 4.30×10~7 CFU,showing that the virulence of the strain was 1.98% of SL1344. The immunoprotective efficacy results showed that the protection rate of the animals infected with Salmonella typhimurium was 50% on the immunization by mutant strain.The serum antibodies peaked 21 st d post-immunization. The complemented strain was no significant difference in the biological characteristics with the parent SL1344 strain,and it basically returned to the wild-type level.The SL1344Δ5'-nucleotidase mutant was constructed successfully,and genetic stability,significantly reduced virulence.The above-mentioned results showed that 5'-nucleotidase plays an important role in regulating the virulence of SL1344,which provide a method for studying the relationship between the 5'-nucleotidase gene of Salmonella typhimurium and its pathogenicity,and lay a foundation for studying its role in Salmonella typhimurium infection.
引文
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[25]BERENDS E T,HORSWILL A R,HASTE N M,et al.Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps[J].Journal of Innate Immunity,2010,2(6):576-586.
[26]RADCLIFF F J,FRASER J D,PROFT T.Vaccination with Streptococcus pyogenes nuclease A stimulates a high antibody response but no protective immunity in a mouse model of infection[J].Journal of Medical Microbiology,2015,204(2):185-191.
[27]DERRE-BOBILLOT A,CORTES-PEREZ N G,YAMAMOTO Y,et al.Nuclease A(Gbs066 1),an extracellular nuclease of Streptococcus agalactiae,attacks the neutrophil extracellular traps and is needed for full virulence[J].Molecular Microbiology,2013,89(3):518-531.
[28]SHAN Q,DWYER M,RAHMAN S,et al.Distinct susceptibilities of corneal Pseudomonas aeruginosa clinical isolates to neutrophil extracellular trapmediated immunity[J].Infection and Immunity,2014,82(10):4135-4143.
[29]张俊峰,张春杰,程相朝,等.猪霍乱沙门菌缺失株ΔcyaC78-1和ΔcrpΔcyaC78-1的构建及其生物学特性比较[J].中国兽医科学,2014,44(8):781-788.ZHANG Jun-feng,ZHANG Chun-jie,CHENG Xiang-chao,et al.Construction of Salmonella choleraesuis mutantΔcya C78-1andΔcrpΔcya C78-1 and comparison of their biological characteristics[J].Chinese Veterinary Science,2014,44(8):781-788.
[30]杨亚东,张春杰,程相朝,等.鼠伤寒沙门菌SL1344株分泌性蛋白K1缺失株的构建及其生物学特性的初步研究[J].中国兽医科学,2017,47(9):1087-1093.YANG Ya-dong,ZHANG Chun-jie,CHENG Xiang-chao,et al.Construction and characterization of the secreted protein K1 gene deleted mutant of Salmonella typhimurium SL1344 strain[J].Chinese Veterinary Science,2017,47(9):215-220.
[2]BHUTTA Z A,THRELFALL J.Addressing the global disease burden of typhoid fever[J].The Journal of the American Medical Association,2009,302(8):898-899.
[3]MATAMOUROS S,MILLER S I.S.typhimurium strategies to resist killing by cationic antimicrobial peptides[J].Biochimica et Biophysica Acta,2015,1848(11):3021-3025.
[4]FOLEY S L,JOHNSON T J,RICKE S C,et al.Salmonella pathogenicity and host adaptation in chicken-associated serovars[J].Microbiology and Molecular Biology Reviews,2013,77(4):582-607.
[5]LIU J,SUN L,LIU W,et al.A nuclease from Streptococcus mutans facilitates biofilm dispersal and escape from killing by neutrophil extracellular traps[J].Frontiers in Cellular and Infection Microbiology,2017,7:97.
[6]WEBER D J,MULLEN G P,MILDVAN A S.Conformation of an enzyme-bound substrate of staphylococcal nuclease as determined by NMR[J].Biochemistry,1991,30(30):7425-7437.
[7]TRAN T M,MACINTYRE A,HAWES M,et al.Escaping under ground nets:Extracellular DNases degrade plant extracellular traps and contribute to virulence of the plant pathogenic bacterium Ralstonia solanacearum[J].PLo S Pathogens,2016,12(6):e1005686.
[8]BINNENKADE L,KREIENBAUM M,THORMANN K M.Characterization of ExeM,an extracellular nuclease of Shewanellaoneidensis MR-1[J].Frontiers in Microbiology,2018,9:1761.
[9]WANG L,WANG Z Q,CUI J.Protein changes in Trichinella spiralis,muscle larvae in vitro induced by bovine bile[J].Veterinary Parasitology,2013,194(2-4):164-167.
[10]TULLSON P C,TERJUNG R L.Adenine nucleotide degradation in striated muscle[J].International Journal of Sports Medicine,1990,2:S47-55.
[11]AIRAS L,NIEMELA J,SALMI M,et al.Differential regulation and function of CD73,a phatidylinositollinked 70-k D adhesion molecule,on lymphocytes and endothelial cells[J].Journal of Cell Biology,1997,136(2):421-431.
[12]RESTA R,YAMASHITA Y,THOMPSON L F.Ecto-enzyme and signaling functions of lymphocyte CD73[J].Immunological Reviews,1998,161:95-109.
[13]MORIWAKI Y,YAMAMOTO T,HIGASHINO K.Enzymes involved in purine metabolism-a review of histochemical localization and functional implications[J].Histology and Histopathology,1999,14(4):1321-1340.
[14]DAI J,LAI L,TANG H,et al.Streptococcus suis synthesizes deoxyadenosine and adenosine by 5’-nucleotidase to dampen host immune responses[J].Virulence,2018,9(1):1509-1520.
[15]AGBOR T A,MCCORMICK B A.Salmonella effectors:important players modulating host cell function during infection[J].Cellular Microbiology,201l,13(12):1858-1869.
[16]LATHROP S K,BINDER K A,STARR T,et al.Replication of Salmonella enterica serovar typhimurium in human monocyte-derived macrophages[J].Infection and Immunity,2015,83(7):2661-2671.
[17]KIM J M,CHOE M H,ASAITHAMBI K,et al.Helicobacter pylori HP0425 targets the nucleus with DNase I-like activity[J].Helicobacter,2016,21(3):218-225.
[18]SANCHEZ M,COLOM F.Extracellular DNase activity of Cryp tococcus neoformans and Cryptococcus gattii[J].Revista Iberoamericana de Micologia,2010,27(1):10-13.
[19]DE PAULA MENEZES R,DE MELO RICETOB,BORGES A S,et al.Evaluation of virulence factors of Candida albicans isolated from HIV-positive individuals using HAART[J].Archives of Oral Biology,2016,66:61-65.
[20]GUIMARAES-COSTA A B,DES OUZA-VIEIRA T S,PALET-TA-SILVA R,et al.3'-nucleotidase/nuclease activity allows Lei shmania parasites to escape killing by neutrophil extracellular traps[J].Infection and Immunity,2014,82(4):1732-1740.
[21]STORISTEANU D M,POCOCK J M,COWBURN A S,et al.Evasion of neutrophil extracellular traps by respiratory pathogens[J].American Journal of Respiratory Cell and Molecular Biology,2017,56(4):423-431.
[22]BRINKMANN V,REICHARD U,GOOSMANN C,et al.Neutrophil extracellular traps kill bacteria[J].Science,2004,303(5663):1532-1535.
[23]SUMBY P,BARBIAN K D,GARDNER D J,et al.Extracellular deoxyribonuclease made by group A Streptococcusassists pathogenesis by enhancing evasion of the innate immune response[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(5):1679-1684.
[24]BUCHANAN J T,SIMPSON A J,AZIZ R K,et al.DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps[J].Current Bbiology,2006,16(4):396-400.
[25]BERENDS E T,HORSWILL A R,HASTE N M,et al.Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps[J].Journal of Innate Immunity,2010,2(6):576-586.
[26]RADCLIFF F J,FRASER J D,PROFT T.Vaccination with Streptococcus pyogenes nuclease A stimulates a high antibody response but no protective immunity in a mouse model of infection[J].Journal of Medical Microbiology,2015,204(2):185-191.
[27]DERRE-BOBILLOT A,CORTES-PEREZ N G,YAMAMOTO Y,et al.Nuclease A(Gbs066 1),an extracellular nuclease of Streptococcus agalactiae,attacks the neutrophil extracellular traps and is needed for full virulence[J].Molecular Microbiology,2013,89(3):518-531.
[28]SHAN Q,DWYER M,RAHMAN S,et al.Distinct susceptibilities of corneal Pseudomonas aeruginosa clinical isolates to neutrophil extracellular trapmediated immunity[J].Infection and Immunity,2014,82(10):4135-4143.
[29]张俊峰,张春杰,程相朝,等.猪霍乱沙门菌缺失株ΔcyaC78-1和ΔcrpΔcyaC78-1的构建及其生物学特性比较[J].中国兽医科学,2014,44(8):781-788.ZHANG Jun-feng,ZHANG Chun-jie,CHENG Xiang-chao,et al.Construction of Salmonella choleraesuis mutantΔcya C78-1andΔcrpΔcya C78-1 and comparison of their biological characteristics[J].Chinese Veterinary Science,2014,44(8):781-788.
[30]杨亚东,张春杰,程相朝,等.鼠伤寒沙门菌SL1344株分泌性蛋白K1缺失株的构建及其生物学特性的初步研究[J].中国兽医科学,2017,47(9):1087-1093.YANG Ya-dong,ZHANG Chun-jie,CHENG Xiang-chao,et al.Construction and characterization of the secreted protein K1 gene deleted mutant of Salmonella typhimurium SL1344 strain[J].Chinese Veterinary Science,2017,47(9):215-220.