2型猪链球菌stk/stp1基因敲除株的构建及特性研究
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摘要
目的丝/苏氨酸激酶(STK)与丝/苏氨酸磷酸酶(STP)通过对蛋白质的可逆磷酸化调控原核生物的各项生理活动。文中旨在研究2型猪链球菌中国强毒株05ZYH33中、stk/stp1同时缺失对其生物学特性及致病性的影响。方法运用同源重组的方法构建2型猪链球菌中国强毒株05ZYH33的stk/stp1基因敲除突变株Δstk/stp1,比较分析野毒株05ZYH33与突变株Δstk/stp1的生物学特性;通过细胞黏附实验、抗吞噬实验以及小鼠感染模型分析stk/stp1缺失对细菌毒力的影响。将30只SPF级BALB/c小鼠,用随机数字表法分成05ZYH33组(注射1 m L野毒株菌液)、Δstk/stp1组(注射1 m L突变株菌液)和THB组(注射1 m L的新鲜THB液体培养基),每组10只。结果 PCR结果显示,stk/stp1基因被壮观霉素抗性基因Spcr替换,突变株构建成功;生物学特性实验表明,05ZYH33和Δstk/stp1在接种2 h后开始缓慢增殖,在7 h时都到达平台期。突变株进入对数生长期(A600≈0.4)的时间比野毒株推迟1 h左右,到达平台期后的菌体密度比野毒株低(0.8 vs 1.0)。05ZYH33和Δstk/stp1在血平板上均长出灰白色、圆形半透明、湿润、表面光滑的细小菌落。菌落周围有明显的β-溶血环,菌落形态和溶血活性无明显变化。小鼠致病性实验显示,05ZYH33组小鼠在12 h内全部死亡,Δstk/stp1组小鼠在12 h内死亡9只,24 h内全部死亡,THB组无发病和死亡症状。结论 stk/stp1同时缺失可能主要影响细菌增殖、分裂相关蛋白的调控。
Objective Serine/threonine kinases( STK) and phosphatases( STP) regulate various physiological activities of prokaryotes by reversible phosphorylation of proteins. This paper aimed to study the effects of simultaneous deletion of the stk and stp1 genes on the biological characteristics and pathogenicity of streptococcus suis type 2,the Chinese virulent strain 05 ZYH33. Methods The double mutant of the stk and stp1 genes of 05 ZYH33 was constructed by homologous recombination. The biological characteristics of the wild strain 05 ZYH33 and the mutant strain Δstk/stp1 were compared. The effects of the stk and stp1 deletion on bacterial virulence was analyzed using cell adhesion assay,anti-phagocytosis assay and the mouse model of infection. Results RT-PCR showed that the stk and stp1 genes were replaced by the spectinomycin resistance gene Spcrand the mutant strain was successfully constructed. Experi-ments of biological characterization revealed gradually increased value of 05 ZYH33 and Δstk/stp1 at 2 hours after inoculation and a plateau period at 7 hours. The logarithmic phase of the mutant strain( A600≈0.4) was 1 hour later than that of the wild one,and the bacterial density of the former was lower than that of the latter after the plateau period( 0.8 vs 1. 0). On the blood plates of 05 ZYH33 and Δstk/stp1 were observed greyish,round,semitransparent,wet and smooth-surfaced tiny bacterial colonies,around which there were hemolysis rings with no significant differences in colony morphology and hemolytic activity. In the experiment on pathogenicity,the mice of the 05 ZYH33 group all died within 12 hours while 9 of the 30 mice in the Δstk/stp1 group died within 12 hours and all died within 24 hours.Conclusion The simultaneous deletion of the stk and stp1 genes may mainly affect the regulation of the proteins associated with bacterial proliferation and division.
Objective Serine/threonine kinases( STK) and phosphatases( STP) regulate various physiological activities of prokaryotes by reversible phosphorylation of proteins. This paper aimed to study the effects of simultaneous deletion of the stk and stp1 genes on the biological characteristics and pathogenicity of streptococcus suis type 2,the Chinese virulent strain 05 ZYH33. Methods The double mutant of the stk and stp1 genes of 05 ZYH33 was constructed by homologous recombination. The biological characteristics of the wild strain 05 ZYH33 and the mutant strain Δstk/stp1 were compared. The effects of the stk and stp1 deletion on bacterial virulence was analyzed using cell adhesion assay,anti-phagocytosis assay and the mouse model of infection. Results RT-PCR showed that the stk and stp1 genes were replaced by the spectinomycin resistance gene Spcrand the mutant strain was successfully constructed. Experi-ments of biological characterization revealed gradually increased value of 05 ZYH33 and Δstk/stp1 at 2 hours after inoculation and a plateau period at 7 hours. The logarithmic phase of the mutant strain( A600≈0.4) was 1 hour later than that of the wild one,and the bacterial density of the former was lower than that of the latter after the plateau period( 0.8 vs 1. 0). On the blood plates of 05 ZYH33 and Δstk/stp1 were observed greyish,round,semitransparent,wet and smooth-surfaced tiny bacterial colonies,around which there were hemolysis rings with no significant differences in colony morphology and hemolytic activity. In the experiment on pathogenicity,the mice of the 05 ZYH33 group all died within 12 hours while 9 of the 30 mice in the Δstk/stp1 group died within 12 hours and all died within 24 hours.Conclusion The simultaneous deletion of the stk and stp1 genes may mainly affect the regulation of the proteins associated with bacterial proliferation and division.
引文
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[14]Rajagopal L,Clancy A,Rubens CE,et al.A Eukaryotic Type Serine/Threonine Kinase and Phosphatase in Streptococcus agalactiae Reversibly Phosphorylate an Inorganic Pyrophosphatase and Affect Growth,Cell Segregation,and Virulence[J].Biol Chem,2003,278(16):14429-14441.
[15]Pancholi V,Boel G,Jin H,et al.Streptococcus pyogenes Ser/Thr kinase-regulated cell wall hydrolase is a cell division planerecognizing and chain-forming virulence factor[J].J Biol Chem,2010,285(40):30861-30874.
[16]Echenique J,Kadioglu A,Romao S,et al.Protein serine/threonine kinase Stk P positively controls virulence and competence in Streptococcus pneumoniae[J].Infect Immun,2004,72(4):2434-2437.
[17]杜骁杰,郭静静,王晶,等.2型猪链球菌丝氨酸/苏氨酸磷酸酶重组蛋白的制备及鉴定[J].医学研究生学报,2014,27(2):120-123.
[18]Zhu H,Huang D,Zhang W,et al.The novel virulence-related genestp of Streptococcus suis serotype 9 strain contributes to a significant reduction in mouse mortality[J].Microb Pathog,2011,51(6):442-453.
[19]Shakir SM,Bryant KM,Larabee JL,et al.Regulatory Interactions of a Virulence-Associated Serine/Threonine PhosphataseKinase Pair in Bacillus anthracis[J].J Bacteriol,2010,192(2):400-409.
[20]Wei F,Huang HY,Zhong NJ,et al.A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation,Genetic Competence,and Acid Resistance in Streptococcus mutans[J].J Bacteriol,2006,188(4):1628-1632.
[2]Tang J,Wang C,Feng Y,et al.2006.Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2[J].PLo S Med,2006,5(3):0068-0076.
[3]钟璟皓,胡丹,唐慧娴,等.2型猪链球菌SSU05_0736基因敲除株的构建及其特性分析[J].医学研究生学报,2016,29(6):571-576.
[4]Feng Y,Zhang H,Wu Z,et al.Streptococcus suis infection:an emerging/reemerging challenge of bacterial infectious diseases?[J]Virulence,2014,5(4):477-497.
[5]Liu Z,Zheng H,Gottschalk M,et al.Development of Multiplex PCR Assays for the Identification of the 33 Serotypes of Streptococcus suis[J].PLo S One,2013,8(8):e72070.63-69.
[6]Nomoto R,Maruyama F,Ishida S,et al.Reappraisal of the taxonomy of Streptococcus suis serotypes 20,22 and 26:Streptococcus parasuis sp.nov[J].Int J Syst Evol Microbiol,2015,65(2):438-443.
[7]Pereira SF,Goss L,Dworkin J.Eukaryote-like serine/threonine kinases and phosphatases in bacteria[J].Microbiol Mol Biol Rev,2011,75(1):192-212.
[8]Chen C,Tang J,Dong W,et al.A Glimpse of Streptococcal Toxic Shock Syndrome from Comparative Genomics of S.suis 2 Chinese Isolates[J].PLo S One,2007,2(3):e315.
[9]杜骁杰.猪链球菌真核样丝氨酸/苏氨酸激酶stk基因功能的实验研究[D].南京:南京师范大学生命科学学院,2014:1-69.
[10]杜骁杰,王玲,郑峰,等.猪链球菌真核样丝氨酸/苏氨酸激酶stk基因敲除株的构建[J].中国人兽共患病学报,2013,29(12):1162-1167.
[11]杜骁杰,倪华,王玲,等.2型猪链球菌丝氨酸/苏氨酸激酶对细菌生物学特性的影响[J].中国人兽共患病学报,2014,30(11):1166-1168.
[12]Hytonen J,Haataja S,Finne J.Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells:investigation of the possible role of surface pullulanase and cysteine protease,and the transcriptional regulator Rgg[J].BMC Microbiol,2006,6(1):18.
[13]Munoz-Dorado J,Inouye S,Inouye M.A gene encoding a protein serine/threonine kinase is required for normal development of M.xanthus,a gram-negative bacterium[J].Cell,1991,67(5):995-1006.
[14]Rajagopal L,Clancy A,Rubens CE,et al.A Eukaryotic Type Serine/Threonine Kinase and Phosphatase in Streptococcus agalactiae Reversibly Phosphorylate an Inorganic Pyrophosphatase and Affect Growth,Cell Segregation,and Virulence[J].Biol Chem,2003,278(16):14429-14441.
[15]Pancholi V,Boel G,Jin H,et al.Streptococcus pyogenes Ser/Thr kinase-regulated cell wall hydrolase is a cell division planerecognizing and chain-forming virulence factor[J].J Biol Chem,2010,285(40):30861-30874.
[16]Echenique J,Kadioglu A,Romao S,et al.Protein serine/threonine kinase Stk P positively controls virulence and competence in Streptococcus pneumoniae[J].Infect Immun,2004,72(4):2434-2437.
[17]杜骁杰,郭静静,王晶,等.2型猪链球菌丝氨酸/苏氨酸磷酸酶重组蛋白的制备及鉴定[J].医学研究生学报,2014,27(2):120-123.
[18]Zhu H,Huang D,Zhang W,et al.The novel virulence-related genestp of Streptococcus suis serotype 9 strain contributes to a significant reduction in mouse mortality[J].Microb Pathog,2011,51(6):442-453.
[19]Shakir SM,Bryant KM,Larabee JL,et al.Regulatory Interactions of a Virulence-Associated Serine/Threonine PhosphataseKinase Pair in Bacillus anthracis[J].J Bacteriol,2010,192(2):400-409.
[20]Wei F,Huang HY,Zhong NJ,et al.A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation,Genetic Competence,and Acid Resistance in Streptococcus mutans[J].J Bacteriol,2006,188(4):1628-1632.