猪链球菌2型05ZYH33菌株启动子的筛选和鉴定
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摘要
为了筛选和鉴定用于猪链球菌(S.suis)致病因子和致病机制研究的启动子,本研究通过质谱鉴定了来自S.suis 2型05ZYH33菌株的5个高丰度蛋白质,并通过其驱动GFP蛋白和甘露糖苷酶SSU05_1921的表达水平,评估了这5个蛋白相应的基因启动子的强弱。结果显示,这5种蛋白质分别为SSU05_0177、SSU05_0530、SSU05_1503、SSU05_1815和SSU05_1868,相应的5个启动子P_(0177)、P_(0530)、P_(1503)、P_(1815)和P_(1868)均能够有效驱动GFP在E.coli和S.suis中的高水平表达,但是P_(1815)、P_(0177)和P_(1868)不能驱动SSU05_1921在S.suis中的表达。P_(0530)和P_(1503)能够驱动SSU05_1921在S.suis中的表达,并且P_(1503)启动子驱动GFP和SSU05_1921的表达水平均比P_(0530)启动子驱动的高两倍以上。因此,P_(0530)和P_(1503)是S.suis的强启动子,并且P_(1503)更强于P_(0530)。这在高水平表达GFP用于标记菌体,以及构建无启动子基因的回复突变菌株中发挥关键作用,在S.suis遗传操作中具有很好的应用前景。
To screen and identify promoters for the study of virulence factors and pathogenic mechanisms of Streptococcus suis(S.suis), five high-abundance proteins were identified by MALDI-TOF/TOF-MS from S.suis serotype 2 strain 05 ZYH33, and the corresponding gene promoters of these 5 proteins were evaluated by their capability of driving protein expression of GFP and a mannosidase SSU05_1921. Results revealed that the five proteins were SSU05_0177, SSU05_0530, SSU05_1503, SSU05_1815,and SSU05_1868, respectively, and the corresponding promoters P_(0177), P_(0530), P_(1503), P_(1815) and P_(1868) could effectively drive the expression of GFP in E.coli and S.suis, but P_(1815), P_(0177) and P_(1868) could not drive SSU05_1921 expression in S.suis. In contrast, P_(0530) and P_(1503) were able to drive expression of SSU05_1921 in S.suis, and the latter showed more than twice higher efficiency than P_(0530) in driving both GFP and SSU05_1921 expression in S.suis. Therefore, both P_(0530) and P_(1503) are strong promoters of S.suis. P_(1503) is even stronger than P_(0530). These two strong promoters are playing a key role in expressing GFP at high levels for labeling cells and constructing back mutations without promoter genes, and it is promising for genetic manipulation and pathological study in S.suis.
To screen and identify promoters for the study of virulence factors and pathogenic mechanisms of Streptococcus suis(S.suis), five high-abundance proteins were identified by MALDI-TOF/TOF-MS from S.suis serotype 2 strain 05 ZYH33, and the corresponding gene promoters of these 5 proteins were evaluated by their capability of driving protein expression of GFP and a mannosidase SSU05_1921. Results revealed that the five proteins were SSU05_0177, SSU05_0530, SSU05_1503, SSU05_1815,and SSU05_1868, respectively, and the corresponding promoters P_(0177), P_(0530), P_(1503), P_(1815) and P_(1868) could effectively drive the expression of GFP in E.coli and S.suis, but P_(1815), P_(0177) and P_(1868) could not drive SSU05_1921 expression in S.suis. In contrast, P_(0530) and P_(1503) were able to drive expression of SSU05_1921 in S.suis, and the latter showed more than twice higher efficiency than P_(0530) in driving both GFP and SSU05_1921 expression in S.suis. Therefore, both P_(0530) and P_(1503) are strong promoters of S.suis. P_(1503) is even stronger than P_(0530). These two strong promoters are playing a key role in expressing GFP at high levels for labeling cells and constructing back mutations without promoter genes, and it is promising for genetic manipulation and pathological study in S.suis.
引文
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[13]Yu Yan-fei,Wang Hon-gen,Wang Jia,et al.Elongation factor thermo unstable(EF-Tu)moonlights as an adhesin on the surface of Mycoplasma hyopneumoniae by binding to fibronectin[J].Front Microbiol,2018,9:974.
[14]Esgleas M,Li Y,Hancock M A,et al.Isolation and characterization of alpha-enolase,a novel fibronectin-binding protein from Streptococcus suis[J].Microbiology,2008,154(Pt9):2668-2679.
[2]Berthelot-Herault F,Marois C,Gottschalk M,et al.Genetic diversity of Streptococcus suis strains isolated from pigs and humans as revealed by pulsed-field gel electrophoresis[J].J Clin Microbiol,2002,40(2):615-619.
[3]Du Peng-cheng,Zheng Han,Zhou Jie-ping,et al.Detection of multiple parallel transmission outbreak of Streptococcus suis human infection by use of genome epidemiology,China,2005[J].Emerg Infect Dis,2017,23(2):204-211.
[4]Henriques M X,Catalao M J,Figueiredo J,et al.Construction of improved tools for protein localization studies in Streptococcus pneumoniae[J].PLoS One,2013,8(1):e55049.
[5]Pinas G E,Reinoso-Vizcaino N M,Yandar Barahona N Y,et al.Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae[J].PLoS Pathog,2018,14(6):e1007118.
[6]Koczula A,Willenborg J,Bertram R,et al.Establishment of a cre recombinase based mutagenesis protocol for markerless gene deletion in Streptococcus suis[J].J Microbiol Methods,2014,107:80-83.
[7]Chen Chen,Tang Jia-qi,Dong Wei,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.
[8]Zaccaria E,van Baarlen P,de Greeff A,et al.Control of competence for DNA transformation in Streptococcus suis by genetically transferable pherotypes[J].PLo S One,2014,9(6):e99394.
[9]Robb M,Hobbs J K,Woodiga S A,et al.Molecular characterization of N-glycan degradation and transport in Streptococcus pneumoniae and its contribution to virulence[J].PLo S Pathog,2017,13(1):e1006090.
[10]Yu Jie,Pian Ya-ya,Ge Jing-peng,et al.Functional and structural characterization of the antiphagocytic properties of a novel transglutaminase from Streptococcus suis[J].J Biol Chem,2015,290(31):19081-19092.
[11]Pyburn T M,Bensing B A,Xiong Y Q,et al.A structural model for binding of the serine-rich repeat adhesin Gsp B to host carbohydrate receptors[J].PLo S Pathog,2011,7(7):e1002112.
[12]Detmers F J,Lanfermeijer F C,Abele R,et al.Combinatorial peptide libraries reveal the ligand-binding mechanism of the oligopeptide receptor OppA of Lactococcus lactis[J].Proc Natl Acad Sci USA,2000,97(23):12487-12492.
[13]Yu Yan-fei,Wang Hon-gen,Wang Jia,et al.Elongation factor thermo unstable(EF-Tu)moonlights as an adhesin on the surface of Mycoplasma hyopneumoniae by binding to fibronectin[J].Front Microbiol,2018,9:974.
[14]Esgleas M,Li Y,Hancock M A,et al.Isolation and characterization of alpha-enolase,a novel fibronectin-binding protein from Streptococcus suis[J].Microbiology,2008,154(Pt9):2668-2679.