肺炎链球菌LytR蛋白影响细菌生长和磷壁酸的合成
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摘要
为了初步明确lytR基因的保守性以及探讨LytR蛋白在肺炎链球菌中的生物学功能。本研究利用PCR和测序方法分析lytR基因20种不同序列型肺炎链球菌临床分离株的保守性;利用Western blotting技术分析lytR在细菌不同生长阶段的表达情况;随后,利用红霉素片段替代lytR基因构建D39ΔlytR缺陷菌株,通过生长曲线分析、电镜下形态观察、以及磷壁酸含量测定揭示LytR蛋白在细菌生长、细菌形态以及磷壁酸合成中的作用。研究发现20种不同序列型肺炎链球菌临床菌株的lytR基因序列一致性>99.3%;Western blotting结果显示lytR在细菌不同生长阶段稳定表达;D39ΔlytR缺陷株生长明显变缓,细菌表现为不成熟自溶;D39ΔlytR全菌磷壁酸含量显著减少,而培养基上清中的磷壁酸含量显著增多。本研究表明lytR基因非常保守,在肺炎链球菌不同生长阶段均有表达,并可能通过连接酶的作用影响细菌生长和参与肺炎链球菌磷壁酸的合成,有望成为一种新的抗生素和药物靶点。
To clarify the conservation of the lytR gene and its roles in pneumococcal growth and biosynthesis of teichoic acids. The conservation of lytR gene was examined in 20 sequence types(STs) of pneumococcal clinical isolates by PCR and was further examined by sequencing; the expression of LytR protein at different growth stages was examined by Western blotting; subsequently, D39ΔlytR mutant was constructed by replacing the lytR gene with anerm cassette. The role of lytR in the growthmorphology was determined by analyzing the bacterial growth profile. The effects of lytR on pneumococal morphology and the biosynthesis of teichoic acids, were analyzed by TEM and immunoblotting strategies. The lytR gene was conserved in the tested 20 STs of pneumococcal clinical isolates, with>99.3% gene identity. lytR was constitutively expressed at different growth stages shown by Western Blotting. D39ΔlytR displayed a significant growth defect and exhibited premature autolysis. Importantly,D39ΔlytR mutant showed a significantly reduce in the amount of whole cell teichoic acids, while an increase level in the supernatant of the mutant was observed relative to WT D39 strain. lytR gene is expressed conservatively and constantly in different growth stages in pneumococcal strains. lytR might be involved in bacterial growth and the biosynthesis of pneumococcal teichoic acids by acting through the action of the ligase. The present study providesevidence that LytR may be a promising antibiotic candidate and a new drug target for clinical prevention and treatment.
To clarify the conservation of the lytR gene and its roles in pneumococcal growth and biosynthesis of teichoic acids. The conservation of lytR gene was examined in 20 sequence types(STs) of pneumococcal clinical isolates by PCR and was further examined by sequencing; the expression of LytR protein at different growth stages was examined by Western blotting; subsequently, D39ΔlytR mutant was constructed by replacing the lytR gene with anerm cassette. The role of lytR in the growthmorphology was determined by analyzing the bacterial growth profile. The effects of lytR on pneumococal morphology and the biosynthesis of teichoic acids, were analyzed by TEM and immunoblotting strategies. The lytR gene was conserved in the tested 20 STs of pneumococcal clinical isolates, with>99.3% gene identity. lytR was constitutively expressed at different growth stages shown by Western Blotting. D39ΔlytR displayed a significant growth defect and exhibited premature autolysis. Importantly,D39ΔlytR mutant showed a significantly reduce in the amount of whole cell teichoic acids, while an increase level in the supernatant of the mutant was observed relative to WT D39 strain. lytR gene is expressed conservatively and constantly in different growth stages in pneumococcal strains. lytR might be involved in bacterial growth and the biosynthesis of pneumococcal teichoic acids by acting through the action of the ligase. The present study providesevidence that LytR may be a promising antibiotic candidate and a new drug target for clinical prevention and treatment.
引文
Brunskill E.W.,and Bayles K.W.,1996,Identification of LytSR-regulated genes from Staphylococcus aureus,J.Bacteriol.,178(19):5810-5812
Chan Y.G.,Frankel M.B.,Dengler V.,Schneewind O.,and Missiakas D.,2013,Staphylococcus aureus mutants lacking the Lyt R-CpsA-Psr family of enzymes release cell wall teichoic acids into the extracellular medium,J.Bacteriol.,195(20):4650-4659
Chan Y.G.,Kim H.K.,Schneewind O.,and Missiakas D.,2014,The capsular polysaccharide of Staphylococcus aureus is attached to peptidoglycan by the Lyt R-CpsA-Psr(LCP)family of enzymes,J.Biol.Chem.,289(22):15680-15690
Chatfield C.H.,Koo H.,and Quivey R.G.,2005,The putative autolysin regulator Lyt R in Staphylococcus aureus plays a role in cell division and is growth-phase regulated,Microbiol.,151(2):625-631
Dai J.,2015,The optimalization of preparation and transformation of E.coli competence cell,Jiangsu Nongye Kexue(Jiangsu Agricultural Sciences),43(4):53-54(代军,2015,大肠杆菌感受态细胞制备及转化条件优化,江苏农业科学,43(4):53-54)
Eberhardt A.,Hoyland C.N.,Vollmer D.,Bisle S.,Cleverley R.M.,Johnsborg O.,Havarstein L.S.,Lewis R.J.,and Vollmer W.,2012,Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae,Microb.Drug.Resist.,18(3):240-255
Kawai Y.,Marles-Wright J.,Cleverley R.M.,Emmins R.,Ishikawa S.,Kuwano M.,Heinz N.,Bui N.K.,Hoyland C.N.,Ogasawara N.,Lewis R.J.,Vollmer W.,Daniel R.A.,and Errington J.,2011,A widespread family of bacterial cell wall assembly proteins,EMBO J.,30(24):4931-4941
Lazarevic V.,Margot P.,Soldo B.,and Karamata D.,1992,Sequencing and analysis of the Bacillus subtilis lyt RABC divergon:a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier,J.Gen.Microbiol.,138(9):1949-1961
Liu Y.,Wang H.,Chen M.,Sun Z.,Zhao R.,Zhang L.,Wang H.,Zhang H.,Wang L.,Chu Y.,Liu Y.,and Ni Y.,2008,Serotype distribution and antimicrobial resistance patterns of Streptococcus pneumoniae isolated from children in China younger than 5 years,Diag.Microbiol.Infect.Dis.,61(3):256-263
Mellroth P.,Daniels R.,Eberhardt A.,Ronnlund D.,Blom H.,Widengren J.,Normark S.,and Henriques-Normark B.,2012,Lyt A,major autolysin of Streptococcus pneumoniae,requires access to nascent peptidoglycan,J.Bio.Chem.,287(14):11018-11029
Min X.,Zhong W.,Zhao S.,Dong J.,Dong S.,Zhou A.,Yan W.,and Wang D.,2013,Studies on expression,purification,crystal growth and optimization of putative transcription factor Lyt R from Streptococcus pneumoniae,Shengwu Yixue Gongchengxue Zazhi(Journal of Biomedical Engineering),30(4):812-816(闵迅,钟文,赵沙沙,董杰,董珊珊,周爱娥,闫文娟,汪德强,2013,肺炎链球菌假想转录因子Lyt R的表达、纯化、晶体生长及优化,生物医学工程学杂志,30(4):812-816)
Monterroso B.,Lopez-Zumel C.,Garcia J.L.,Saiz J.L.,Garcia P.,Campillo N.E.,a nd Menendez M.,2005,Unravelling the structure of the pneumococcal autolytic lysozyme,Biochem.J.,391(1):41-49
O'Brien K.L.,Wolfson L.J.,Watt J.P.,Henkle E.,Deloria-Knoll M.,McCall N.,Lee E.,Mulholland K.,and Levine O.S.,2009,Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years:global estimates,Lancet,374(9693):893-902
Raz R.,Elhanan G.,Shimoni Z.,Kitzes R.,Rudnicki C.,Igra Y.,and Yinnon A.,1997,Pneumococcal bacteremia in hospitalized israeli adults:epidemiology and resistance to penicillin,israeli adult pneumococcal bacteremia group,Clin.Infect.Dis.,24(6):1164-1168
Steinmoen H.,Knutsen E.,and Havarstein L.S.,2001,Induction of natural competence in Streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population,Proc.Natl.Acad.Sci.USA,99(11):7681-7686
Tuomanen E.I.,Austrian R.,and Masure H.R.,1995,Pathogenesis of pneumococcal infection,N.Engl.J.Med.,332(19):1280-1284
Wu K.,Huang J.,Zhang Y.,Xu W.,Xu H.,Wang L.,Cao J.,Zhang X.,and Yin Y.,2014,A novel protein,RafX,is important for common cell wall polysaccharide biosynthesis in Streptococcus pneumoniae:Implications for bacterial virulence,J.Bacteriol.,196(18):3324-3334
Wu K.,Zhang W.,Cui Y.,Zhang X.,Xu W.,Pang D.,Liu X.,and Wang H.,2010,Construction and over-expression of a pneumolysin mutant derived from Streptococcus pneumoniae D39,Yixue Fenzishengwuxue Zazhi(Journal of Medical Molecular Biology),7(5):395-400(吴凯峰,张薇薇,崔亚利,张雪梅,胥文春,庞丹,刘鑫,王虹,2010,一种减毒肺炎链球菌溶血素突变体的构建与表达,医学分子生物学杂志,7(5):395-400)
Wu K.,Zhang X.,Shi J.,Li N.,Li D.,Luo M.,Cao J.,Yin N.,Wang H.,Xu W.,He Y.,and Yin Y.,2010,Immunization with a combination of three pneumococcal proteins confers additive and broad protection against Streptococcus pneumoniae infections in mice,Infect.Immun.,78(3):1276-1283
Xue L.,Yao K.,Xie G.,Zheng Y.,Wang C.,Shang Y.,Wang H.,Wan L.,Liu L.,Li C.,Ji W.,Xu X.,Wang Y.,Xu P.,Liu Z.,Yu S.,and Yang Y.,2010,Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates that cause invasive disease among Chinese children,Clin.Infect.Dis.,50(5):741-744
Chan Y.G.,Frankel M.B.,Dengler V.,Schneewind O.,and Missiakas D.,2013,Staphylococcus aureus mutants lacking the Lyt R-CpsA-Psr family of enzymes release cell wall teichoic acids into the extracellular medium,J.Bacteriol.,195(20):4650-4659
Chan Y.G.,Kim H.K.,Schneewind O.,and Missiakas D.,2014,The capsular polysaccharide of Staphylococcus aureus is attached to peptidoglycan by the Lyt R-CpsA-Psr(LCP)family of enzymes,J.Biol.Chem.,289(22):15680-15690
Chatfield C.H.,Koo H.,and Quivey R.G.,2005,The putative autolysin regulator Lyt R in Staphylococcus aureus plays a role in cell division and is growth-phase regulated,Microbiol.,151(2):625-631
Dai J.,2015,The optimalization of preparation and transformation of E.coli competence cell,Jiangsu Nongye Kexue(Jiangsu Agricultural Sciences),43(4):53-54(代军,2015,大肠杆菌感受态细胞制备及转化条件优化,江苏农业科学,43(4):53-54)
Eberhardt A.,Hoyland C.N.,Vollmer D.,Bisle S.,Cleverley R.M.,Johnsborg O.,Havarstein L.S.,Lewis R.J.,and Vollmer W.,2012,Attachment of capsular polysaccharide to the cell wall in Streptococcus pneumoniae,Microb.Drug.Resist.,18(3):240-255
Kawai Y.,Marles-Wright J.,Cleverley R.M.,Emmins R.,Ishikawa S.,Kuwano M.,Heinz N.,Bui N.K.,Hoyland C.N.,Ogasawara N.,Lewis R.J.,Vollmer W.,Daniel R.A.,and Errington J.,2011,A widespread family of bacterial cell wall assembly proteins,EMBO J.,30(24):4931-4941
Lazarevic V.,Margot P.,Soldo B.,and Karamata D.,1992,Sequencing and analysis of the Bacillus subtilis lyt RABC divergon:a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier,J.Gen.Microbiol.,138(9):1949-1961
Liu Y.,Wang H.,Chen M.,Sun Z.,Zhao R.,Zhang L.,Wang H.,Zhang H.,Wang L.,Chu Y.,Liu Y.,and Ni Y.,2008,Serotype distribution and antimicrobial resistance patterns of Streptococcus pneumoniae isolated from children in China younger than 5 years,Diag.Microbiol.Infect.Dis.,61(3):256-263
Mellroth P.,Daniels R.,Eberhardt A.,Ronnlund D.,Blom H.,Widengren J.,Normark S.,and Henriques-Normark B.,2012,Lyt A,major autolysin of Streptococcus pneumoniae,requires access to nascent peptidoglycan,J.Bio.Chem.,287(14):11018-11029
Min X.,Zhong W.,Zhao S.,Dong J.,Dong S.,Zhou A.,Yan W.,and Wang D.,2013,Studies on expression,purification,crystal growth and optimization of putative transcription factor Lyt R from Streptococcus pneumoniae,Shengwu Yixue Gongchengxue Zazhi(Journal of Biomedical Engineering),30(4):812-816(闵迅,钟文,赵沙沙,董杰,董珊珊,周爱娥,闫文娟,汪德强,2013,肺炎链球菌假想转录因子Lyt R的表达、纯化、晶体生长及优化,生物医学工程学杂志,30(4):812-816)
Monterroso B.,Lopez-Zumel C.,Garcia J.L.,Saiz J.L.,Garcia P.,Campillo N.E.,a nd Menendez M.,2005,Unravelling the structure of the pneumococcal autolytic lysozyme,Biochem.J.,391(1):41-49
O'Brien K.L.,Wolfson L.J.,Watt J.P.,Henkle E.,Deloria-Knoll M.,McCall N.,Lee E.,Mulholland K.,and Levine O.S.,2009,Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years:global estimates,Lancet,374(9693):893-902
Raz R.,Elhanan G.,Shimoni Z.,Kitzes R.,Rudnicki C.,Igra Y.,and Yinnon A.,1997,Pneumococcal bacteremia in hospitalized israeli adults:epidemiology and resistance to penicillin,israeli adult pneumococcal bacteremia group,Clin.Infect.Dis.,24(6):1164-1168
Steinmoen H.,Knutsen E.,and Havarstein L.S.,2001,Induction of natural competence in Streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population,Proc.Natl.Acad.Sci.USA,99(11):7681-7686
Tuomanen E.I.,Austrian R.,and Masure H.R.,1995,Pathogenesis of pneumococcal infection,N.Engl.J.Med.,332(19):1280-1284
Wu K.,Huang J.,Zhang Y.,Xu W.,Xu H.,Wang L.,Cao J.,Zhang X.,and Yin Y.,2014,A novel protein,RafX,is important for common cell wall polysaccharide biosynthesis in Streptococcus pneumoniae:Implications for bacterial virulence,J.Bacteriol.,196(18):3324-3334
Wu K.,Zhang W.,Cui Y.,Zhang X.,Xu W.,Pang D.,Liu X.,and Wang H.,2010,Construction and over-expression of a pneumolysin mutant derived from Streptococcus pneumoniae D39,Yixue Fenzishengwuxue Zazhi(Journal of Medical Molecular Biology),7(5):395-400(吴凯峰,张薇薇,崔亚利,张雪梅,胥文春,庞丹,刘鑫,王虹,2010,一种减毒肺炎链球菌溶血素突变体的构建与表达,医学分子生物学杂志,7(5):395-400)
Wu K.,Zhang X.,Shi J.,Li N.,Li D.,Luo M.,Cao J.,Yin N.,Wang H.,Xu W.,He Y.,and Yin Y.,2010,Immunization with a combination of three pneumococcal proteins confers additive and broad protection against Streptococcus pneumoniae infections in mice,Infect.Immun.,78(3):1276-1283
Xue L.,Yao K.,Xie G.,Zheng Y.,Wang C.,Shang Y.,Wang H.,Wan L.,Liu L.,Li C.,Ji W.,Xu X.,Wang Y.,Xu P.,Liu Z.,Yu S.,and Yang Y.,2010,Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates that cause invasive disease among Chinese children,Clin.Infect.Dis.,50(5):741-744
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