锌离子对肺炎链球菌影响的蛋白质组学研究
摘要
肺炎链球菌细菌为革兰氏阳性致病菌,能产生多种毒素,可引起急性咽炎、呼吸道感染、丹毒、脓疱病、软组织感染、心内膜炎和脑膜炎等,产毒株还可引起猩红热。由于该细菌的强大毒力性及对抗生素的抗性,这种感染的预防和治疗成为困扰国际医学界的一大难题。锌离子对于人体非常重要,人体中的锌离子浓度介于几微摩到几百摩尔之间。金属锌离子对人体的免疫系统起重要的作用,锌离子的缺失会导致机体免疫力下降。锌离子对于细菌的生长代谢是非常重要的,他们通过与相应的蛋白结合,尤其是与生物代谢催化剂-酶结合,共同在细菌体内发挥生物作用。金属锌离子对于细菌的粘附力与毒力有重要影响。因此,对锌离子在细菌的作用的研究尤为重要。
目的：建立有无锌离子培养条件下的肺炎链球菌全蛋白质和锌结合蛋白质双向电泳图谱,结合质谱技术鉴定并分析差异表达蛋白,并结合生物信息手段对蛋白质相互关系进行分析,揭示锌离子对肺炎链球菌的生长代谢的调控机制,从而为预防和治疗肺炎链球菌感染提供理论基础。
方法：本研究利用不同梯度的锌离子浓度处理肺炎链球菌,收集菌体并提取总蛋白。结合金属亲和层析技术,利用双向凝胶电泳技术分离蛋白质,建立锌调控蛋白差异表达双向凝胶电泳图谱,经ImageMsater图像分析软件识别分析差异表达蛋白质斑点后,切取差异蛋白质斑点胶块,胶内酶解后再用基质辅助激光解析电离飞行时间质谱仪(MALDI-TOF-MS)进行分析,数据库查询后对差异表达的蛋白质点进行鉴定,结合生物信息手段进行分析,以期找出锌离子调控的肺炎链球菌致病相关的蛋白。
结果：1.建立了有无锌离子作用的锌离子调控蛋白双向凝胶电泳图谱,鉴定了96个差异表达蛋白斑点,共67个差异蛋白。其中32个表达下调,35个表达上调。锌离子调控蛋白的作用主要体现在氧化还原作用、帮助蛋白质折叠和糖代谢等方面。建立了有无锌离子作用的锌结合蛋白的双向电泳图谱,鉴定了10个差异表达蛋白斑点,共7个差异蛋白,其中在无锌离子培养条件下1个下调表达,6个上调表达。
结论：本实验建立了肺炎链球菌蛋白双向电泳技术,通过分析有无锌离子处理肺炎链球菌蛋白的表达变化,对锌离子调控蛋白作用机制有了一定的了解。锌离子调控蛋白对肺炎链球菌的作用主要体现在氧化还原反应、蛋白质折叠、金属转运等方面。结合金属亲和层析技术研究锌离子结合蛋白,鉴定出在无锌离子条件下肺炎链球菌差异表达蛋白,研究结果为进一步探索肺炎链球菌的致病机制提供了理论依据,为相应的药物研发奠定了基础。
The human pathogen Streptococcus pneumoniae is a commensal of the nasopharynx, but it can become virulent by infecting the lung, middle ear, brain, and bloodstream, causing severe diseases such as pneumonia, otitis media, meningitis, and sepsis. An important metal ion that S. pneumoniae could face is Zn2+, which is present in the human body in concentrations ranging from a fewμM to over 100μM. In the host, Zn2+is of great importance for immunity, as it is necessary for proper functioning of immune cells, and mild Zn2+ deficiency severely affects immune function. Zn2+is crucial for bacterial growth and metabolism as it is the co-factor in many key enzymes. Moreover, Zn2+is involved in bacterial adherence and virulence. This project aims to investigate the molecular basis of the bacterial growth inhibition induced by Zn2+ deficiency and to find out the mechanism of Zn2+effects on S. pnemunoniae virulence.
AIM:Establish the 2DE maps of Zn2+regulatory proteins of S. pnemunoniae and investigate the molecular mechanism of Zn2+-regulated growth inhibition of S. pneumoniae.
METHOD:2DE-based proteomic approach was used to compare the differences of protein expression in S. pnemunoniae with and without zinc ion.2DE-gel maps were established for the total proteins extracted from the bacteria and the sub-proteome after Zn-IMAC separation, respectively. Proteins with altered expression were identified by MALDI-TOF/TOF mass spectrometry and further characterized based on their molecular functions.
RESULT:With the 2DE analysis for the total protein extracts,96 protein spots were found to have different expressions with Zn2+deficiency and 67 unique proteins including 32 down-regulated and 35 up-regulated proteins were identified. These Zn2+regulatory proteins involve in various biological processes including oxidation, protein folding and carbohydrate metabolism. By using Zn-IMAC separation, Zn2+binding proteins in S. pneumoniae were also profiled by 2DE and 10 different protein spots with 7 unique proteins were identified.
CONCLUSION:Zn2+deficiency affects the growth of S. pneumoniae. Zn2+regulated proteins can be identified by 2DE-based proteomic technology. Further functional analysis revealed that these Zn2+regulated proteins involve in oxidation, proteins folding and carbohydrate metabolism. These results provide information to help us better understand the molecular basis of the growth and virulence inhibition of S. pneumoniae induced by Zn2+ deficiency.
目的：建立有无锌离子培养条件下的肺炎链球菌全蛋白质和锌结合蛋白质双向电泳图谱,结合质谱技术鉴定并分析差异表达蛋白,并结合生物信息手段对蛋白质相互关系进行分析,揭示锌离子对肺炎链球菌的生长代谢的调控机制,从而为预防和治疗肺炎链球菌感染提供理论基础。
方法：本研究利用不同梯度的锌离子浓度处理肺炎链球菌,收集菌体并提取总蛋白。结合金属亲和层析技术,利用双向凝胶电泳技术分离蛋白质,建立锌调控蛋白差异表达双向凝胶电泳图谱,经ImageMsater图像分析软件识别分析差异表达蛋白质斑点后,切取差异蛋白质斑点胶块,胶内酶解后再用基质辅助激光解析电离飞行时间质谱仪(MALDI-TOF-MS)进行分析,数据库查询后对差异表达的蛋白质点进行鉴定,结合生物信息手段进行分析,以期找出锌离子调控的肺炎链球菌致病相关的蛋白。
结果：1.建立了有无锌离子作用的锌离子调控蛋白双向凝胶电泳图谱,鉴定了96个差异表达蛋白斑点,共67个差异蛋白。其中32个表达下调,35个表达上调。锌离子调控蛋白的作用主要体现在氧化还原作用、帮助蛋白质折叠和糖代谢等方面。建立了有无锌离子作用的锌结合蛋白的双向电泳图谱,鉴定了10个差异表达蛋白斑点,共7个差异蛋白,其中在无锌离子培养条件下1个下调表达,6个上调表达。
结论：本实验建立了肺炎链球菌蛋白双向电泳技术,通过分析有无锌离子处理肺炎链球菌蛋白的表达变化,对锌离子调控蛋白作用机制有了一定的了解。锌离子调控蛋白对肺炎链球菌的作用主要体现在氧化还原反应、蛋白质折叠、金属转运等方面。结合金属亲和层析技术研究锌离子结合蛋白,鉴定出在无锌离子条件下肺炎链球菌差异表达蛋白,研究结果为进一步探索肺炎链球菌的致病机制提供了理论依据,为相应的药物研发奠定了基础。
The human pathogen Streptococcus pneumoniae is a commensal of the nasopharynx, but it can become virulent by infecting the lung, middle ear, brain, and bloodstream, causing severe diseases such as pneumonia, otitis media, meningitis, and sepsis. An important metal ion that S. pneumoniae could face is Zn2+, which is present in the human body in concentrations ranging from a fewμM to over 100μM. In the host, Zn2+is of great importance for immunity, as it is necessary for proper functioning of immune cells, and mild Zn2+ deficiency severely affects immune function. Zn2+is crucial for bacterial growth and metabolism as it is the co-factor in many key enzymes. Moreover, Zn2+is involved in bacterial adherence and virulence. This project aims to investigate the molecular basis of the bacterial growth inhibition induced by Zn2+ deficiency and to find out the mechanism of Zn2+effects on S. pnemunoniae virulence.
AIM:Establish the 2DE maps of Zn2+regulatory proteins of S. pnemunoniae and investigate the molecular mechanism of Zn2+-regulated growth inhibition of S. pneumoniae.
METHOD:2DE-based proteomic approach was used to compare the differences of protein expression in S. pnemunoniae with and without zinc ion.2DE-gel maps were established for the total proteins extracted from the bacteria and the sub-proteome after Zn-IMAC separation, respectively. Proteins with altered expression were identified by MALDI-TOF/TOF mass spectrometry and further characterized based on their molecular functions.
RESULT:With the 2DE analysis for the total protein extracts,96 protein spots were found to have different expressions with Zn2+deficiency and 67 unique proteins including 32 down-regulated and 35 up-regulated proteins were identified. These Zn2+regulatory proteins involve in various biological processes including oxidation, protein folding and carbohydrate metabolism. By using Zn-IMAC separation, Zn2+binding proteins in S. pneumoniae were also profiled by 2DE and 10 different protein spots with 7 unique proteins were identified.
CONCLUSION:Zn2+deficiency affects the growth of S. pneumoniae. Zn2+regulated proteins can be identified by 2DE-based proteomic technology. Further functional analysis revealed that these Zn2+regulated proteins involve in oxidation, proteins folding and carbohydrate metabolism. These results provide information to help us better understand the molecular basis of the growth and virulence inhibition of S. pneumoniae induced by Zn2+ deficiency.
引文
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2. Lee, M.R., et al., Proteomic analysis of protein expression in Streptococcus pneumoniae in response to temperature shift. J Microbiol,2006.44(4):p.375-82.
3. Bae, S.M., et al., The effect of protein expression of Streptococcus pneumoniae by blood. J Biochem Mol Biol,2006.39(6):p.703-8.
4. Nanduri, B., et al., Quantitative analysis of Streptococcus pneumoniae TIGR4 response to in vitro iron restriction by 2-D LC ESI MS/MS. Proteomics,2008.8(10):p.2104-14.
5. Sun, X., et al., Phosphoproteomic analysis reveals the multiple roles of phosphorylation in pathogenic bacterium Streptococcus pneumoniae. J Proteome Res.9(1):p.275-82.
6. Blencowe, D.K. and A.P. Morby, Zn(II) metabolism in prokaryotes. FEMS Microbiol Rev, 2003.27(2-3):p.291-311.
7. Finney, L.A. and T.V. O'Halloran, Transition metal speciation in the cell:insights from the chemistry of metal ion receptors. Science,2003.300(5621)
8. Moore, C.M. and J.D. Helmann, Metal ion homeostasis in Bacillus subtilis. Curr Opin Microbiol,2005.8(2):p.188-95.
9. Nies, D.H., Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev, 2003.27(2-3):p.313-39.
10. Versieck, J., Trace elements in human body fluids and tissues. Crit Rev Clin Lab Sci, 1985.22(2):p.97-184.
11. Ibs, K.H. and L. Rink, Zinc-altered immune function. J Nutr,2003.133(5 Suppl 1):p. 1452S-6S.
12. Strand, T.A., et al., Pneumococcal pulmonary infection, septicaemia and survival in young zinc-depleted mice. Br J Nutr,2001.86(2):p.301-6.
13. Rolerson, E., et al., The SloR/Dlg metalloregulator modulates Streptococcus mutans virulence gene expression. J Bacteriol,2006.188(14):p.5033-44.
14. Panina, E.M., A.A. Mironov, and M.S. Gelfand, Comparative genomics of bacterial zinc regulons:enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins. Proc Natl Acad Sci U S A,2003.100(17):p.9912-7.
15. Paik, S., et al., The sloABCR operon of Streptococcus mutans encodes an Mn and Fe transport system required for endocarditis virulence and its Mn-dependent repressor. J Bacteriol,2003.185(20):p.5967-75.
16. Montanez, G.E., M.N. Neely, and Z. Eichenbaum, The streptococcal iron uptake (Siu) transporter is required for iron uptake and virulence in a zebrafish infection model. Microbiology,2005.151(Pt 11):p.3749-57.
17. Mitrakul, K., et al., Mutational analysis of the adcCBA genes in Streptococcus gordonii biofilm formation. Oral Microbiol Immunol,2005.20(2):p.122-7.
18. McAllister, L.J., et al., Molecular analysis of the psa permease complex of Streptococcus pneumoniae. Mol Microbiol,2004.53(3):p.889-901.
19. Loo, C.Y., et al., Involvement of the adc operon and manganese homeostasis in Streptococcus gordonii biofilm formation. J Bacteriol,2003.185(9):p.2887-900.
20. Kolenbrander, P.E., et al., The adhesion-associated sca operon in Streptococcus gordonii encodes an inducible high-affinity ABC transporter for Mn2+ uptake. J Bacteriol,1998. 180(2):p.290-5.
21. Kloosterman, T.G., et al., The novel transcriptional regulator SczA mediates protection against Zn2+ stress by activation of the Zn2+ -resistance gene czcD in Streptococcus pneumoniae. Mol Microbiol,2007.65(4):p.1049-63.
22. Johnston, J.W., et al., Lipoprotein PsaA in virulence of Streptococcus pneumoniae: surface accessibility and role in protection from superoxide. Infect Immun,2004.72(10): p.5858-67.
23. Dintilhac, A., et al., Competence and virulence of Streptococcus pneumoniae:Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metalpermeases. Mol Microbiol,1997.25(4):p.727-39.
24. Brown, J.S., et al., Characterization of pit, a Streptococcus pneumoniae iron uptake ABC transporter. Infect Immun,2002.70(8):p.4389-98.
25. Brown, J.S., S.M. Gilliland, and D.W. Holden, A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence. Mol Microbiol,2001.40(3):p.572-85.
26. Ando, M., et al., Characterization of the role of the divalent metal ion-dependent transcriptional repressor MntR in the virulence of Staphylococcus aureus. Infect Immun, 2003.71(5):p.2584-90.
27. Kloosterman, T.G., et al., Opposite effects of Mn2+ and Zn2+ on PsaR-mediated expression of the virulence genes pcp A, prtA, and psaBCA of Streptococcus pneumoniae. J Bacteriol, 2008.190(15):p.5382-93.
28. Fey, S.J. and P.M. Larsen,2D or not 2D. Two-dimensional gel electrophoresis. Curr Opin Chem Biol,2001.5(1):p.26-33.
29. Adams, L.D. and S.R. Gallagher, Two-dimensional gel electrophoresis. Curr Protoc Immunol,2005. Chapter 8:p. Unit 85.
30. Ge, F., et al., Proteomic and functional analyses reveal a dual molecular mechanism underlying arsenic-induced apoptosis in human multiple myeloma cells. J Proteome Res, 2009.8(6):p.3006-19.
31. O'Farrell, P.H., High resolution two-dimensional electrophoresis of proteins. J Biol Chem, 1975.250(10):p.4007-21.
32. Aebersold, R. and M. Mann, Mass spectrometry-based proteomics. Nature,2003. 422(6928):p.198-207.
33. Blaha, G., R.E. Stanley, and T.A. Steitz, Formation of the first peptide bond:the structure of EF-P bound to the 70S ribosome. Science,2009.325(5943):p.966-70.
34. Bailly, M. and V. de Crecy-Lagard, Predicting the pathway involved in post-translational modification of Elongation factor P in a subset of bacterial species. Biol Direct.5:p.3.
35. Defeu Soufo, H.J., et al., Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein. Proc Natl Acad Sci U S A.107(7):p.3163-8.
36. Moll, I., et al., Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli. Mol Microbiol,2002.44(5):p.1387-96.
37. Moazed, D. and H.F. Noller, Intermediate states in the movement of transfer RNA in the ribosome. Nature,1989.342(6246):p.142-8.
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2. Lee, M.R., et al., Proteomic analysis of protein expression in Streptococcus pneumoniae in response to temperature shift. J Microbiol,2006.44(4):p.375-82.
3. Bae, S.M., et al., The effect of protein expression of Streptococcus pneumoniae by blood. J Biochem Mol Biol,2006.39(6):p.703-8.
4. Nanduri, B., et al., Quantitative analysis of Streptococcus pneumoniae TIGR4 response to in vitro iron restriction by 2-D LC ESI MS/MS. Proteomics,2008.8(10):p.2104-14.
5. Sun, X., et al., Phosphoproteomic analysis reveals the multiple roles of phosphorylation in pathogenic bacterium Streptococcus pneumoniae. J Proteome Res.9(1):p.275-82.
6. Blencowe, D.K. and A.P. Morby, Zn(II) metabolism in prokaryotes. FEMS Microbiol Rev, 2003.27(2-3):p.291-311.
7. Finney, L.A. and T.V. O'Halloran, Transition metal speciation in the cell:insights from the chemistry of metal ion receptors. Science,2003.300(5621)
8. Moore, C.M. and J.D. Helmann, Metal ion homeostasis in Bacillus subtilis. Curr Opin Microbiol,2005.8(2):p.188-95.
9. Nies, D.H., Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev, 2003.27(2-3):p.313-39.
10. Versieck, J., Trace elements in human body fluids and tissues. Crit Rev Clin Lab Sci, 1985.22(2):p.97-184.
11. Ibs, K.H. and L. Rink, Zinc-altered immune function. J Nutr,2003.133(5 Suppl 1):p. 1452S-6S.
12. Strand, T.A., et al., Pneumococcal pulmonary infection, septicaemia and survival in young zinc-depleted mice. Br J Nutr,2001.86(2):p.301-6.
13. Rolerson, E., et al., The SloR/Dlg metalloregulator modulates Streptococcus mutans virulence gene expression. J Bacteriol,2006.188(14):p.5033-44.
14. Panina, E.M., A.A. Mironov, and M.S. Gelfand, Comparative genomics of bacterial zinc regulons:enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins. Proc Natl Acad Sci U S A,2003.100(17):p.9912-7.
15. Paik, S., et al., The sloABCR operon of Streptococcus mutans encodes an Mn and Fe transport system required for endocarditis virulence and its Mn-dependent repressor. J Bacteriol,2003.185(20):p.5967-75.
16. Montanez, G.E., M.N. Neely, and Z. Eichenbaum, The streptococcal iron uptake (Siu) transporter is required for iron uptake and virulence in a zebrafish infection model. Microbiology,2005.151(Pt 11):p.3749-57.
17. Mitrakul, K., et al., Mutational analysis of the adcCBA genes in Streptococcus gordonii biofilm formation. Oral Microbiol Immunol,2005.20(2):p.122-7.
18. McAllister, L.J., et al., Molecular analysis of the psa permease complex of Streptococcus pneumoniae. Mol Microbiol,2004.53(3):p.889-901.
19. Loo, C.Y., et al., Involvement of the adc operon and manganese homeostasis in Streptococcus gordonii biofilm formation. J Bacteriol,2003.185(9):p.2887-900.
20. Kolenbrander, P.E., et al., The adhesion-associated sca operon in Streptococcus gordonii encodes an inducible high-affinity ABC transporter for Mn2+ uptake. J Bacteriol,1998. 180(2):p.290-5.
21. Kloosterman, T.G., et al., The novel transcriptional regulator SczA mediates protection against Zn2+ stress by activation of the Zn2+ -resistance gene czcD in Streptococcus pneumoniae. Mol Microbiol,2007.65(4):p.1049-63.
22. Johnston, J.W., et al., Lipoprotein PsaA in virulence of Streptococcus pneumoniae: surface accessibility and role in protection from superoxide. Infect Immun,2004.72(10): p.5858-67.
23. Dintilhac, A., et al., Competence and virulence of Streptococcus pneumoniae:Adc and PsaA mutants exhibit a requirement for Zn and Mn resulting from inactivation of putative ABC metalpermeases. Mol Microbiol,1997.25(4):p.727-39.
24. Brown, J.S., et al., Characterization of pit, a Streptococcus pneumoniae iron uptake ABC transporter. Infect Immun,2002.70(8):p.4389-98.
25. Brown, J.S., S.M. Gilliland, and D.W. Holden, A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence. Mol Microbiol,2001.40(3):p.572-85.
26. Ando, M., et al., Characterization of the role of the divalent metal ion-dependent transcriptional repressor MntR in the virulence of Staphylococcus aureus. Infect Immun, 2003.71(5):p.2584-90.
27. Kloosterman, T.G., et al., Opposite effects of Mn2+ and Zn2+ on PsaR-mediated expression of the virulence genes pcp A, prtA, and psaBCA of Streptococcus pneumoniae. J Bacteriol, 2008.190(15):p.5382-93.
28. Fey, S.J. and P.M. Larsen,2D or not 2D. Two-dimensional gel electrophoresis. Curr Opin Chem Biol,2001.5(1):p.26-33.
29. Adams, L.D. and S.R. Gallagher, Two-dimensional gel electrophoresis. Curr Protoc Immunol,2005. Chapter 8:p. Unit 85.
30. Ge, F., et al., Proteomic and functional analyses reveal a dual molecular mechanism underlying arsenic-induced apoptosis in human multiple myeloma cells. J Proteome Res, 2009.8(6):p.3006-19.
31. O'Farrell, P.H., High resolution two-dimensional electrophoresis of proteins. J Biol Chem, 1975.250(10):p.4007-21.
32. Aebersold, R. and M. Mann, Mass spectrometry-based proteomics. Nature,2003. 422(6928):p.198-207.
33. Blaha, G., R.E. Stanley, and T.A. Steitz, Formation of the first peptide bond:the structure of EF-P bound to the 70S ribosome. Science,2009.325(5943):p.966-70.
34. Bailly, M. and V. de Crecy-Lagard, Predicting the pathway involved in post-translational modification of Elongation factor P in a subset of bacterial species. Biol Direct.5:p.3.
35. Defeu Soufo, H.J., et al., Bacterial translation elongation factor EF-Tu interacts and colocalizes with actin-like MreB protein. Proc Natl Acad Sci U S A.107(7):p.3163-8.
36. Moll, I., et al., Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli. Mol Microbiol,2002.44(5):p.1387-96.
37. Moazed, D. and H.F. Noller, Intermediate states in the movement of transfer RNA in the ribosome. Nature,1989.342(6246):p.142-8.
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