肺炎克雷伯菌KP1＿4563基因突变株的构建及其生物膜表型分析
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摘要
KP1_4563基因是肺炎克雷伯菌NTUH-K2044中假设的蛋白编码基因,与Ⅲ型菌毛的功能有关。本实验首先采用同源重组基因敲除方法构建肺炎克雷伯菌KP1_4563基因缺失的突变株(Kp-△4563),然后PCR扩增KP1_4563基因片段,克隆到质粒p BAD33上,将重组质粒导入Kp-△4563获得回补株(Kpc-△4563)。分别测定野生株、突变株、回补株用普通LB培养基,改良Minka培养基以及含胆汁盐的LB培养基培养时生物膜形成能力,以此来探讨KP1_4563基因以及不同培养基对肺炎克雷伯菌体外生物膜形成的影响。我们成功构建KP1_4563基因缺失的突变株和回补株Kpc-△4563。与野生株相比,突变株Kp-△4563生物膜形成能力减弱,回补株介于野生株和突变株之间。使用改良Minka培养基使菌株菌毛化以及加入胆汁盐可以增加生物膜的形成能力。这些分析表明肺炎克雷伯菌KP1_4563基因能正调控细菌生物膜的形成。体外培养使细菌菌毛化以及加入胆汁盐可以促进生物膜的形成。
KP1_4563 of K. pneumoniae NTUH-K2044 is a hypothetical protein-encoding gene, and is related to the function of type Ⅲ fimbria. Firstly, the KP1_4563 gene deletion mutant of Klebsiella pneumoniae(Kp-△4563) was constructed by homologous recombination gene knock-out method. Then, the KP1_4563 gene fragment was amplified and cloned into plasmid p BAD33, and then the recombinant plasmid was transferred into Kp-△4563 to construct the complemented strain(Kpc-△4563). The biofilm formation ability of wild strain, mutant strain, complemented strain which were cultured with LB medium or Minka medium or LB medium with 1.0% bile salt were tested respectively,to determine the effects of KP1_4563 gene and different culture mediums on biofilm formation ability of Klebsiella pneumoniae. In this study, we constructed the KP1_4563-deletion mutant and complemented strain Kpc-△4563 successfully. Compared with wild strain, the biofilm formation ability of mutant KP1_4563 decreased while that of complemented strain Kpc-△4563 was between the wild starin and the mutant strain. The biofilm formation ability of strains could be increased by using modified Minka medium to fimbriate the strains and by adding bile salt. These results indicated that the KP1_4563 gene of Klebsiella pneumoniae could positively regulates biofilm formation and the biofilm formation could be promoted by culture in vitro to fimbriate the strains and by the addition of bile salts.
KP1_4563 of K. pneumoniae NTUH-K2044 is a hypothetical protein-encoding gene, and is related to the function of type Ⅲ fimbria. Firstly, the KP1_4563 gene deletion mutant of Klebsiella pneumoniae(Kp-△4563) was constructed by homologous recombination gene knock-out method. Then, the KP1_4563 gene fragment was amplified and cloned into plasmid p BAD33, and then the recombinant plasmid was transferred into Kp-△4563 to construct the complemented strain(Kpc-△4563). The biofilm formation ability of wild strain, mutant strain, complemented strain which were cultured with LB medium or Minka medium or LB medium with 1.0% bile salt were tested respectively,to determine the effects of KP1_4563 gene and different culture mediums on biofilm formation ability of Klebsiella pneumoniae. In this study, we constructed the KP1_4563-deletion mutant and complemented strain Kpc-△4563 successfully. Compared with wild strain, the biofilm formation ability of mutant KP1_4563 decreased while that of complemented strain Kpc-△4563 was between the wild starin and the mutant strain. The biofilm formation ability of strains could be increased by using modified Minka medium to fimbriate the strains and by adding bile salt. These results indicated that the KP1_4563 gene of Klebsiella pneumoniae could positively regulates biofilm formation and the biofilm formation could be promoted by culture in vitro to fimbriate the strains and by the addition of bile salts.
引文
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Fang C.T.,Chuang Y.P.,Shun C.T.,Chang S.C.,and Wang J.T.,2004,A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications,Journal of Experimental Medicine,199(5):697-705
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He L.Y.,Han W.Y.,Jia Y.,Lei L.C.,and Yang Y.,2007,Typing of fmibriae of avian Klebsiella pneumoniae,Zhongguo Shengwu Zhipinxue Zazhi(China Journal of Biologicals),20(8):575-577(何礼洋,韩文瑜,贾艳,雷连成,杨洋,2007,鸡源肺炎克雷伯菌菌毛的分型,中国生物制品学杂志,20(8):575-577)
Hung D.T.,Zhu J.,Sturtevant D.,and Mekalanos J.J.,2006,Bile acids stimulate biofilm formation in Vibrio cholera,Molecular Microbiology,59(1):193-201
Ko W.C.,Paterson D.L.,Sagnimeni A.J.,Hansen D.S.,Gottberg A.V.,Mohapatra S.,Casellas J.M.,Goossens H.,Mulazimoglu L.,Trenholme G.,Klugman K.P.,McCormack J.G.,and Yu V.L.,2002,Community-acquired Klebsiella pneumoniae bacteremia:global differences in clinical patterns,Emerging Infectious Diseases,8(2):160-166
Langley J.M.,Hanakowski M.,and Leblanc J.C.,2001,Unique epidemiology of nosocomial urinary tract infection in children,American Journal of Infection Control,29(2):94-98
Leid J.G.,Willson C.J.,Shirtliff M.E.,Hassett D.J.,Parsek M.R.,and Jeffers A.K.,2005,The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing,Journal of Immunology,175(11):7512-7518
Liao P.F.,Nie W.,Yu Y.X.,Tong P.G.,Li S.B.,and Zhu Y.L.,2016,ZFNs,TALENs and CRISPR-Cas targeted genome editing technologies and their applications in plants,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(10):1062-1071(廖鹏飞,聂旺,余雅心,童普国,李绍波,朱有林,2016,ZFNs、TALENs和CRISPR-Cas基因组靶向编辑技术及其在植物中的应用,分子植物育种,14(10):1062-1071)
Luo M.,Yang S.,Li X.,Liu P.,Xue J.,Zhou X.,Su K.,Xu X.,Qing Y.,Qiu J.,and Li Y.,2017,The KP1_4563 gene is regulated by the c AMP receptor protein and controls type 3 fimbrial function in Klebsiella pneumoniae NTUH-K2044,PLoSOne,12(7):1-14
Marschall J.,Fraser V.J.,Doherty J.,and Warren D.K.,2009,Between community and hospital:healthcare-associated gramnegative bacteremia among hospitalized patients,Infection Control and Hospital Epidemiology,30(11):1050-1056
Pumbwe L.,Skilbeck C.A.,Nakano V.,Avila-Campos M.J.,Piazza R.M.,and Wexler H.M.,2007,Bile salts enhance bacterial co-aggregation,bacterial-intestinal epithelial cell adhesion,biofilm formation and antimicrobial resistance of Bacteroides fragilis,Microb Pathogenesis,43(2-3):78-87
Sharma S.,Mohan H.,Sharma S.,and Chhibber S.,2011,A comparative study of induction of pneumonia in mice with planktonic and biofilm cells of Klebsiella pneumoniae,Microbiology and Immunology,55(5):295-303
Sligl W.,Taylor G.,and Brindley P.G.,2006,Five years of nosocomial Gram-negative bacteremia in a general intensive care unit:epidemiology,antimicrobial susceptibility patterns,and outcomes,International Journal of Infectious Diseases,10(4):320-325
Stahlhut S.G.,Struve C.,Krogfelt K.A.,and Reisner A.,2012,Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae,Pathogens and Disease,65(2):350-359
Xu L.,and Li B.,2016,Advances in the study of the mechanism of biofilm formation by Klebsiella pneumoniae.Zhongguo Bingyuan Weishengwuxue Zazhi(Journal of Pathogen Biology),11(11):1056(徐丽,李蓓,2016,肺炎克雷伯菌生物膜形成机制的研究进展,中国病原微生物学杂志,11(11):1056)
Zheng Y.,Liu X.P.,and Liu J.H.,2007,Highly efficient transfor mation with plasmid DNA in Klebsiella pneumoniae,Weishengwu Xuebao(Acta Microbiological Sinica),47(4):721-724(郑艳,刘喜朋,刘建华,2007,外源载体高效转化肺炎克雷伯菌的新途径,微生物学报,47(4):721-724)
Chen K.M.,Chiang M.K.,Wang M.,Ho H.C.,Lu M.C.,and Lai Y.C.,2014,The role of pga C in Klebsiella pneumoniae virulence and biofilm formation,Microb Pathogenesis,77:89-99
Fang C.T.,Chuang Y.P.,Shun C.T.,Chang S.C.,and Wang J.T.,2004,A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications,Journal of Experimental Medicine,199(5):697-705
Gao Y.S,Feng T.,Qiu J.F.,Yang R.F.,Zhou D.S.,and Li Y.L.,2013,A method for constructing unmarked deletion mutants of Klebsiella pneumoniae,Junshi Yixue(Military Medical Sciences),37(6):431-434(高钰双,冯甜,邱景富,杨瑞馥,周冬生,李迎丽,2013,肺炎克雷伯菌基因无痕敲除方法的建立,军事医学,37(6):431-434)
He L.Y.,Han W.Y.,Jia Y.,Lei L.C.,and Yang Y.,2007,Typing of fmibriae of avian Klebsiella pneumoniae,Zhongguo Shengwu Zhipinxue Zazhi(China Journal of Biologicals),20(8):575-577(何礼洋,韩文瑜,贾艳,雷连成,杨洋,2007,鸡源肺炎克雷伯菌菌毛的分型,中国生物制品学杂志,20(8):575-577)
Hung D.T.,Zhu J.,Sturtevant D.,and Mekalanos J.J.,2006,Bile acids stimulate biofilm formation in Vibrio cholera,Molecular Microbiology,59(1):193-201
Ko W.C.,Paterson D.L.,Sagnimeni A.J.,Hansen D.S.,Gottberg A.V.,Mohapatra S.,Casellas J.M.,Goossens H.,Mulazimoglu L.,Trenholme G.,Klugman K.P.,McCormack J.G.,and Yu V.L.,2002,Community-acquired Klebsiella pneumoniae bacteremia:global differences in clinical patterns,Emerging Infectious Diseases,8(2):160-166
Langley J.M.,Hanakowski M.,and Leblanc J.C.,2001,Unique epidemiology of nosocomial urinary tract infection in children,American Journal of Infection Control,29(2):94-98
Leid J.G.,Willson C.J.,Shirtliff M.E.,Hassett D.J.,Parsek M.R.,and Jeffers A.K.,2005,The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing,Journal of Immunology,175(11):7512-7518
Liao P.F.,Nie W.,Yu Y.X.,Tong P.G.,Li S.B.,and Zhu Y.L.,2016,ZFNs,TALENs and CRISPR-Cas targeted genome editing technologies and their applications in plants,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(10):1062-1071(廖鹏飞,聂旺,余雅心,童普国,李绍波,朱有林,2016,ZFNs、TALENs和CRISPR-Cas基因组靶向编辑技术及其在植物中的应用,分子植物育种,14(10):1062-1071)
Luo M.,Yang S.,Li X.,Liu P.,Xue J.,Zhou X.,Su K.,Xu X.,Qing Y.,Qiu J.,and Li Y.,2017,The KP1_4563 gene is regulated by the c AMP receptor protein and controls type 3 fimbrial function in Klebsiella pneumoniae NTUH-K2044,PLoSOne,12(7):1-14
Marschall J.,Fraser V.J.,Doherty J.,and Warren D.K.,2009,Between community and hospital:healthcare-associated gramnegative bacteremia among hospitalized patients,Infection Control and Hospital Epidemiology,30(11):1050-1056
Pumbwe L.,Skilbeck C.A.,Nakano V.,Avila-Campos M.J.,Piazza R.M.,and Wexler H.M.,2007,Bile salts enhance bacterial co-aggregation,bacterial-intestinal epithelial cell adhesion,biofilm formation and antimicrobial resistance of Bacteroides fragilis,Microb Pathogenesis,43(2-3):78-87
Sharma S.,Mohan H.,Sharma S.,and Chhibber S.,2011,A comparative study of induction of pneumonia in mice with planktonic and biofilm cells of Klebsiella pneumoniae,Microbiology and Immunology,55(5):295-303
Sligl W.,Taylor G.,and Brindley P.G.,2006,Five years of nosocomial Gram-negative bacteremia in a general intensive care unit:epidemiology,antimicrobial susceptibility patterns,and outcomes,International Journal of Infectious Diseases,10(4):320-325
Stahlhut S.G.,Struve C.,Krogfelt K.A.,and Reisner A.,2012,Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae,Pathogens and Disease,65(2):350-359
Xu L.,and Li B.,2016,Advances in the study of the mechanism of biofilm formation by Klebsiella pneumoniae.Zhongguo Bingyuan Weishengwuxue Zazhi(Journal of Pathogen Biology),11(11):1056(徐丽,李蓓,2016,肺炎克雷伯菌生物膜形成机制的研究进展,中国病原微生物学杂志,11(11):1056)
Zheng Y.,Liu X.P.,and Liu J.H.,2007,Highly efficient transfor mation with plasmid DNA in Klebsiella pneumoniae,Weishengwu Xuebao(Acta Microbiological Sinica),47(4):721-724(郑艳,刘喜朋,刘建华,2007,外源载体高效转化肺炎克雷伯菌的新途径,微生物学报,47(4):721-724)
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