肺炎链球菌自杀性荧光报告质粒的构建与评价
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摘要
肺炎链球菌（S.pneumoniae, S.pn）是一种条件致病菌，自然状态下就具有较高的转化率。在肺炎链球菌的进化、变异和致病过程中环境因素起着相当重要的作用，研究肺炎链球菌的致病机制必须考虑体内环境因素。怎样在体内真实的环境中去分析细菌毒力因子的作用是至关重要的。近几年作为活细胞探针的绿色荧光蛋白为我们提供了一种直观、简便、准确的体内研究手段。将绿色荧光蛋白与体内表达技术结合起来用于研究细菌毒力因子的表达，既可以分析细菌毒力基因的功能又可以筛选出未知的毒力基因。目的：构建肺炎链球菌自杀性荧光报告质粒，将肺炎链球菌溶血素基因与荧光报告基因融合，在体内环境中通过荧光的观察来分析溶血素的表达情况，为进一步研究肺炎链球菌毒力因子在活细胞内的定位和功能分析以及规模筛选毒力基因打下基础。方法：采用基因克隆技术，在肺炎链球菌自杀质粒pEVP3中插入融合基因ply-gfp。利用自杀质粒同源重组的原理，将重组自杀质粒整合入肺炎链球菌染色体中建立稳定的重组菌株。通过对绿色荧光蛋白的直接观察，在体内跟踪肺炎链球菌感染小鼠的过程以及肺炎链球菌溶血素的表达情况。结
果：①成功构建了pEVP3-gfp质粒和pEVP3-ply-gfp质粒，建立了稳定重组gfp的肺炎链球菌菌株。②重组肺炎链球菌与未重组肺炎链球菌在生理活性上没有区别，荧光和溶血素均能正确表达，且荧光在细菌生长的对数后期表达较强。③动物实验表明荧光观察的结果与细菌培养的结果完全一致，同时证实溶血素在细菌自溶后释放到胞外产生毒力作用的活性。结论：利用自杀质粒同源重组的原理，将荧光报告基因融合入肺炎链球菌的染色体中，既可以实时定位观察肺炎链球菌在体内致病的分子过程，又可以通过荧光表达的差异来筛选毒力相关基因。
Streptococcus pneumoniae is the opportunity pathogen which processes transformation more efficiently than other microbes. Therefore, environmental factors are relative to the evolution、variation and pathogenicity of S.pn and we should connect them with the pathogenesis of S.pn. It is important to study S.pn in vivo environment. Priorly, we must find a probe suitable for investigation in vivo. GFP is an ideal probe applied to IVET(in vivo expression technology) because it can be directly detected in vivo. Objective: To use green fluorescent protein to observe pneumococcal adhesion and invasion in vivo and analyze pneumococcal virulence factors. Methods: Gene cloning was used to construct suicide plasmids containing the gfp reporter by which homologous recombination was completed between S.pn DNA and fusion gene
ply-gfp. With the technology of expression in vivo we compared the expression of GFP in vivo with the result of S.pn'culturing in media and analyzed pneumococcal virulence factors. Results: Restriction maps show that the structures of pEVP3-gfp and pEVP3-ply-gfp are exactly the same as anticipated. Further results indicate that the recombinant S.pn is not different from the un- recombinant S.pn and that both GFP and PLY were simultaneously expressed in S.pn. Conclusion: A new plasmid was established as the vector for studying the expression of pneumococcal virulence factors in vivo. It will facilitate analyzing and screening the candidate virulence factors.
果：①成功构建了pEVP3-gfp质粒和pEVP3-ply-gfp质粒，建立了稳定重组gfp的肺炎链球菌菌株。②重组肺炎链球菌与未重组肺炎链球菌在生理活性上没有区别，荧光和溶血素均能正确表达，且荧光在细菌生长的对数后期表达较强。③动物实验表明荧光观察的结果与细菌培养的结果完全一致，同时证实溶血素在细菌自溶后释放到胞外产生毒力作用的活性。结论：利用自杀质粒同源重组的原理，将荧光报告基因融合入肺炎链球菌的染色体中，既可以实时定位观察肺炎链球菌在体内致病的分子过程，又可以通过荧光表达的差异来筛选毒力相关基因。
Streptococcus pneumoniae is the opportunity pathogen which processes transformation more efficiently than other microbes. Therefore, environmental factors are relative to the evolution、variation and pathogenicity of S.pn and we should connect them with the pathogenesis of S.pn. It is important to study S.pn in vivo environment. Priorly, we must find a probe suitable for investigation in vivo. GFP is an ideal probe applied to IVET(in vivo expression technology) because it can be directly detected in vivo. Objective: To use green fluorescent protein to observe pneumococcal adhesion and invasion in vivo and analyze pneumococcal virulence factors. Methods: Gene cloning was used to construct suicide plasmids containing the gfp reporter by which homologous recombination was completed between S.pn DNA and fusion gene
ply-gfp. With the technology of expression in vivo we compared the expression of GFP in vivo with the result of S.pn'culturing in media and analyzed pneumococcal virulence factors. Results: Restriction maps show that the structures of pEVP3-gfp and pEVP3-ply-gfp are exactly the same as anticipated. Further results indicate that the recombinant S.pn is not different from the un- recombinant S.pn and that both GFP and PLY were simultaneously expressed in S.pn. Conclusion: A new plasmid was established as the vector for studying the expression of pneumococcal virulence factors in vivo. It will facilitate analyzing and screening the candidate virulence factors.
引文
1. Hervé Tettelin, Karen E. Nelson, Ian T. Paulsen, etc. Complete Genome Sequence of a Virulent Isolate of Streptococcus pneumoniae Science 2001 293: 498-506.
2. McCullers JA, Tuomanen EI. Molecular pathogenesis of pneumococcal pneumonia. Front Biosci 2001 Aug 1;6:D877-89
3. Varon E. Severe pneumococcal infections: virulence aspects .Arch Pediatr 2001 Sep;8 Suppl 4:752s-756s
4. Henriques Normark B, Normark S. Antibiotic tolerance in pneumococci. Clin Microbiol Infect 2002 Oct;8(10):613-22
5. Regine Hakenbeck.β-lactam-resistant Streptococcus pneumoniae: epidemiology and evolutionary mechanism. Chemotherapy 1999; 45:83-94
6. Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial innovation. Nature 2000 May 18;405(6784):299-304
7. Dowson CG, Barcus V, King S. Horizontal gene transfer and the evolution of resistance and virulence determinants in Streptococcus. Soc Appl Bacteriol Symp Ser 1997;26:42S-51S
8. Schaberg DR. Gene exchange and antimicrobial resistance in gram-positive cocci. Trans Am Clin Climatol Assoc 1996;108:59-66; discussion 66-8
9. Zimmer M. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 2002 Mar;102(3):759-81
10. Rosochacki SJ, Matejczyk M. Green fluorescent protein as a molecular marker in microbiology. Acta Microbiol Pol 2002;51(3):205-16
11. 黄培堂 等译 《分子克隆实验指南》第三版，科学出版社2002，1495-1515
12. Cummings CA, Relman DA. Using DNA microarrays to study host-microbe interactions. Emerg Infect Dis 2000 Sep-Oct;6(5):513-25
13. de Saizieu A, Gardes C, Flint N, etc. Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide. J Bacteriol 2000 Sep;182(17):4696-703
14. 徐建国 《分子医学细菌学》,科学出版社2000，283-292
15. Paton JC, Giammarinaro P. Genome-based analysis of pneumococcal virulence factors: the quest for novel vaccine antigens and drug targets. Trends Microbiol 2001 Nov;9(11):515-8
16. Jean-Pierre Claverys,Agnes Dintilhac,Ekaterina V.Pestova.Construction and evaluation of new drug-resistance cassettes for gene disruption mutagenesis in streptococcus pneumoniae,using an ami test platform.Gene 1995,164:123-128
17. Robert F.Weaver《Molecular Biology》McGraw-Hill Companies,Inc.2000 697-738
18. Tavoularis S, Scazzocchio C, Sophianopoulou V. Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans. Fungal Genet Biol 2001 Jul;33(2):115-25
19. Hennecke, F; Krebber, C; Pluckthun, A. Non-repetitive single-chain Fv linkers selected by selectively infective phage (SIP) technology.
Protein-Eng. 1998 May; 11(5): 405-10
20. Robinson, CR; Sauer, RT. Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. Proc-Natl-Acad-Sci-U-S-A. 1998 May 26; 95(11): 5929-34
21. Skillman LC, Sutherland IW, Jones MV,etc. Green fluorescent protein as a novel species-specific marker in enteric dual-species biofilms. Microbiology 1998 Aug;144 ( Pt 8):2095-101
22. Nieto C, Fernandez de Palencia P, Lopez P,etc. Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae:controlled expression of the green fluorescent protein. Plasmid 2000 May;43(3):205-13
Acebo P, Nieto C, Corrales MA, etc. Quantitative detection of Streptococcus pneumoniae cells harbouring single or multiple copies of
23. the gene encoding the green fluorescent protein. Microbiology 2000 Jun;146 ( Pt 6):1267-73
24. Bartilson M, Marra A, Christine J, etc. Differential fluorescence induction reveals Streptococcus pneumoniae loci regulated by competence stimulatory peptide. Mol Microbiol 2001 Jan;39(1):126-35
25. Marra A, Asundi J, Bartilson M, etc. Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis. Infect Immun 2002 Mar;70(3):1422-33
26. Kadioglu A, Sharpe JA, Lazou I,etc. Use of green fluorescent protein in visualisation of pneumococcal invasion of broncho epithelial cells in vivo. FEMS Microbiol Lett 2001 Jan1; 194(1):105-10
27. Amory-Rivier C, Rieux V, Azoulay-Dupuis E,etc. Selection of virulent mutants of Streptococcus pneumoniae. Utilization of a murine model of septicemia]. Pathol Biol (Paris) 1999 May; 47(5):519-525
28. Polissi A, Pontiggia A, Feger G,etc. Large-scale identification of virulence genes from Streptococcus pneumoniae. Infect Immun 1998 Dec;66(12):5620-9
29. Thanassi JA, Hartman-Neumann SL, Dougherty TJ,etc. Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae. Nucleic Acids Res 2002 Jul 15;30(14):3152-62
30. Hava DL, Camilli A. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol Microbiol 2002 Sep;45(5):1389-406
31. Munoz R, Lopez R, Garcia E. Construction of a new Streptococcus pneumoniae-Escherichia coli shuttle vector based on the replicon of an indigenous pneumococcal cryptic plasmid. Int Microbiol 1999 Mar;2(1):23-8
Lau GW, Haataja S, Lonetto M,etc. A functional genomic analysis of type
32. 3 Streptococcus pneumoniae virulence. Mol Microbiol 2001 May;40(3):555-71
33. Dunny GM, Leonard BA. Cell-cell communication in gram-positive bacteria. Annu Rev Microbiol 1997;51:527-64
34. Dhandayuthapani S, Rasmussen WG, Baseman JB. Identification of mycoplasmal promoters in Escherichia coli using a promoter probe vector with Green Fluorescent Protein as reporter system. Gene 1998 Jul 17;215(1):213-22
35. 35.Davison J. Genetic tools for pseudomonads, rhizobia, and other gram-negative bacteria. Biotechniques 2002 Feb; 32(2):386-8, 390, 392-4,
2. McCullers JA, Tuomanen EI. Molecular pathogenesis of pneumococcal pneumonia. Front Biosci 2001 Aug 1;6:D877-89
3. Varon E. Severe pneumococcal infections: virulence aspects .Arch Pediatr 2001 Sep;8 Suppl 4:752s-756s
4. Henriques Normark B, Normark S. Antibiotic tolerance in pneumococci. Clin Microbiol Infect 2002 Oct;8(10):613-22
5. Regine Hakenbeck.β-lactam-resistant Streptococcus pneumoniae: epidemiology and evolutionary mechanism. Chemotherapy 1999; 45:83-94
6. Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial innovation. Nature 2000 May 18;405(6784):299-304
7. Dowson CG, Barcus V, King S. Horizontal gene transfer and the evolution of resistance and virulence determinants in Streptococcus. Soc Appl Bacteriol Symp Ser 1997;26:42S-51S
8. Schaberg DR. Gene exchange and antimicrobial resistance in gram-positive cocci. Trans Am Clin Climatol Assoc 1996;108:59-66; discussion 66-8
9. Zimmer M. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 2002 Mar;102(3):759-81
10. Rosochacki SJ, Matejczyk M. Green fluorescent protein as a molecular marker in microbiology. Acta Microbiol Pol 2002;51(3):205-16
11. 黄培堂 等译 《分子克隆实验指南》第三版，科学出版社2002，1495-1515
12. Cummings CA, Relman DA. Using DNA microarrays to study host-microbe interactions. Emerg Infect Dis 2000 Sep-Oct;6(5):513-25
13. de Saizieu A, Gardes C, Flint N, etc. Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide. J Bacteriol 2000 Sep;182(17):4696-703
14. 徐建国 《分子医学细菌学》,科学出版社2000，283-292
15. Paton JC, Giammarinaro P. Genome-based analysis of pneumococcal virulence factors: the quest for novel vaccine antigens and drug targets. Trends Microbiol 2001 Nov;9(11):515-8
16. Jean-Pierre Claverys,Agnes Dintilhac,Ekaterina V.Pestova.Construction and evaluation of new drug-resistance cassettes for gene disruption mutagenesis in streptococcus pneumoniae,using an ami test platform.Gene 1995,164:123-128
17. Robert F.Weaver《Molecular Biology》McGraw-Hill Companies,Inc.2000 697-738
18. Tavoularis S, Scazzocchio C, Sophianopoulou V. Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans. Fungal Genet Biol 2001 Jul;33(2):115-25
19. Hennecke, F; Krebber, C; Pluckthun, A. Non-repetitive single-chain Fv linkers selected by selectively infective phage (SIP) technology.
Protein-Eng. 1998 May; 11(5): 405-10
20. Robinson, CR; Sauer, RT. Optimizing the stability of single-chain proteins by linker length and composition mutagenesis. Proc-Natl-Acad-Sci-U-S-A. 1998 May 26; 95(11): 5929-34
21. Skillman LC, Sutherland IW, Jones MV,etc. Green fluorescent protein as a novel species-specific marker in enteric dual-species biofilms. Microbiology 1998 Aug;144 ( Pt 8):2095-101
22. Nieto C, Fernandez de Palencia P, Lopez P,etc. Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae:controlled expression of the green fluorescent protein. Plasmid 2000 May;43(3):205-13
Acebo P, Nieto C, Corrales MA, etc. Quantitative detection of Streptococcus pneumoniae cells harbouring single or multiple copies of
23. the gene encoding the green fluorescent protein. Microbiology 2000 Jun;146 ( Pt 6):1267-73
24. Bartilson M, Marra A, Christine J, etc. Differential fluorescence induction reveals Streptococcus pneumoniae loci regulated by competence stimulatory peptide. Mol Microbiol 2001 Jan;39(1):126-35
25. Marra A, Asundi J, Bartilson M, etc. Differential fluorescence induction analysis of Streptococcus pneumoniae identifies genes involved in pathogenesis. Infect Immun 2002 Mar;70(3):1422-33
26. Kadioglu A, Sharpe JA, Lazou I,etc. Use of green fluorescent protein in visualisation of pneumococcal invasion of broncho epithelial cells in vivo. FEMS Microbiol Lett 2001 Jan1; 194(1):105-10
27. Amory-Rivier C, Rieux V, Azoulay-Dupuis E,etc. Selection of virulent mutants of Streptococcus pneumoniae. Utilization of a murine model of septicemia]. Pathol Biol (Paris) 1999 May; 47(5):519-525
28. Polissi A, Pontiggia A, Feger G,etc. Large-scale identification of virulence genes from Streptococcus pneumoniae. Infect Immun 1998 Dec;66(12):5620-9
29. Thanassi JA, Hartman-Neumann SL, Dougherty TJ,etc. Identification of 113 conserved essential genes using a high-throughput gene disruption system in Streptococcus pneumoniae. Nucleic Acids Res 2002 Jul 15;30(14):3152-62
30. Hava DL, Camilli A. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors. Mol Microbiol 2002 Sep;45(5):1389-406
31. Munoz R, Lopez R, Garcia E. Construction of a new Streptococcus pneumoniae-Escherichia coli shuttle vector based on the replicon of an indigenous pneumococcal cryptic plasmid. Int Microbiol 1999 Mar;2(1):23-8
Lau GW, Haataja S, Lonetto M,etc. A functional genomic analysis of type
32. 3 Streptococcus pneumoniae virulence. Mol Microbiol 2001 May;40(3):555-71
33. Dunny GM, Leonard BA. Cell-cell communication in gram-positive bacteria. Annu Rev Microbiol 1997;51:527-64
34. Dhandayuthapani S, Rasmussen WG, Baseman JB. Identification of mycoplasmal promoters in Escherichia coli using a promoter probe vector with Green Fluorescent Protein as reporter system. Gene 1998 Jul 17;215(1):213-22
35. 35.Davison J. Genetic tools for pseudomonads, rhizobia, and other gram-negative bacteria. Biotechniques 2002 Feb; 32(2):386-8, 390, 392-4,