臺灣博碩士論文加值系統

已知雙分子調控系統RcsB正向調控克雷白氏肺炎桿菌的抗酸反應。rcsB基因的剔除使細菌於低酸鹼值(pH3)之生存率顯著的降低。蛋白質體的研究發現，經過弱酸的適應後rcsB基因剔除使特定蛋白質表現量顯著降低，而後此蛋白質被鑑定為YfdX。位於克雷白氏肺炎桿菌酸適應島嶼的yfdX基因經剔除後，細菌於低酸鹼值的生存率顯著降低。在靜態培養以及弱酸的條件下，剔除rcsB基因或kvhA基因的菌株觀察到PyfdX表現量的降低。本研究中，我們製作ΔrcsBΔyfdX 以及 ΔkvhAΔyfdX之雙突變菌株。抗酸存活率試驗指出ΔkvhAΔyfdX 以及 ΔyfdX 反應相似，然而ΔrcsBΔyfdX 的存活率則相似於ΔrcsB 突變株。顯示酸適應島嶼之雙分子調控系統KvhA在靜態培養條件下的抗酸反應主要透過YfdX表現。目前尚未有研究指出yfdX基因的功能，而使用生物資訊的工具(BLAST, pfam)搜尋指出YfdX具有訊息胜肽但並無其他保守性的序列。酸適應島嶼的伴隨蛋白基因hdeB亦具有訊號序列，且hdeB-hdeD相鄰於yfdX而同樣受到KvhA以及RcsB的調控，於是假設yfdX與hdeB-hdeD在抗酸功能上具有關聯性。我們再進一步製作ΔhdeBΔhdeDΔyfdX以及ΔyfdXΔhdeB1突變菌株，結果顯示ΔhdeBΔhdeD與ΔhdeBΔhdeDΔyfdX有近似的酸存活率;ΔhdeB1基因不具備訊息胜肽ΔyfdX而在抗酸功能未具相關性。接下來，我們建構YfdX表現載體pET30a-yfdX，測試YfdX蛋白質之功能或用於YfdX抗體之製作。體外的伴隨蛋白活性試驗指出His6-YfdX降低ADH的沉澱，His6-YfdX相較於His6-HdeB具有較高的體外伴隨蛋白質活性，但YfdX與HdeB體外的伴隨蛋白活性並沒有明顯的加乘效果。抗體製作完成後，西方點墨法的測試顯示YfdX表現於膜間質，並受靜置培養及弱酸的影響增加表現量。最後，免疫共沉澱的實驗指出在弱酸環境下YfdX能與多數膜間質蛋白質作用，而在中性時與膜間質蛋白質的結合能力較低。以上體外的試驗指出，YfdX在酸性環境下具備伴隨蛋白的活性，保護蛋白質發生沉澱。

The two component system (2CS) response regulator (RR) RscB has been previously shown to play a positive role in the acid-resistance response in Klebsiella pneumoniae CG43. Deletion of rcsB apparently reduced the bacterial survival under pH 3 culture condition. After mild acid adaptation, the proteome study revealed a missing protein spot in the rcsB deletion mutant which later identified as YfdX. Deletion of the yfdX, located on a putative acid fitness island (AFI), appeared to reduce the bacterial survival at pH 3. In statically or mild acidic cultured condition, a reduced level of PyfdX promoter activity in the rcsB deletion and kvhA deletion mutant was also observed. Here, ΔrcsBΔyfdX and ΔkvhAΔyfdX mutants were generated. Acid survival analysis revealed that ΔkvhAΔyfdX and ΔyfdX had similar response to pH 3 treatment, while ΔrcsBΔyfdX revealed same survival level as ΔrcsB mutant. These suggested that KvhA, a 2CS RR encoding gene on the AFI, also plays a regulatory role of acid stress primarily on YfdX expression under statically cultured condition. Furthermore, A signal peptide has been found on YfdX by BLAST but no conserved domain on YfdX protein. We proposed that there are some functional relationships between yfdX and the nearby gene hdeB-hdeD of acid resistance. Therefore, ΔhdeBΔhdeDΔyfdX and ΔyfdXΔhdeB1 mutants were generated and confirmed the correlation between yfdX and hdeB-hdeD under acid stress. Thus, Recombinant YfdX protein has been generated for testing chaperone activity and raising antibodies for YfdX localization detection. Western blotting analysis further confirmed rcsB and kvhA controlling the periplasmic expression of YfdX under acidic and static culture condition. in vitro acid chaperone activity assay revealed the potent protective effects on ADH of YfdX then HdeB. Finally, co-immunoprecipitation test indicated that YfdX can interact with periplasmic proteins under acidic condition.

目錄

中文摘要..........................................................I
Abstract.........................................................II
致謝............................................................III
目錄.............................................................IV
表目錄............................................................V
圖目錄...........................................................VI
縮寫表.........................................................VIII
一、 前言.......................................................1
1.1 克雷白氏肺炎菌.............................................1
1.2 腸胃道病原細菌的抗酸機制...................................2
1.3 分子伴隨蛋白在周邊胞質(Periplasm)的抗酸作用................3
1.4 克雷白氏肺炎桿菌的調控基因與抗酸作用.......................4
二、 材料與方法 ................................................6
三、 結果......................................................11
3.1 yfdX與其鄰近基因對細菌抗酸能力的影響......................11
3.2 YfdX以及HdeB 抗酸能力與伴隨蛋白活性分析...................13
四、 討論......................................................16
相關文獻.........................................................20
表...............................................................25
圖...............................................................29
附錄.............................................................51

1. 林志桓. (2009). RcsB 蛋白質在克雷白氏肺炎桿菌CG43中抗酸能力所扮演的角色.碩士論文,生物醫學研究所,國立交通大學.
2. 曾品瑄. (2011). HdeB、HdeB1、HdeD 蛋白質參與克雷白氏肺炎桿菌CG43抗酸反應的探討.碩士論文,生物醫學研究所,國立交通大學.
3. Beier, D., and R. Gross. 2008. The BvgS/BvgA phosphorelay system of pathogenic Bordetellae: structure, function and evolution. Adv Exp Med Biol 631:149-60.
4. Berg, R. D. 1995. Bacterial translocation from the gastrointestinal tract. Trends Microbiol 3:149-54.
5. Bergholz, T. M., C. L. Tarr, L. M. Christensen, D. J. Betting, and T. S. Whittam. 2007. Recent gene conversions between duplicated glutamate decarboxylase genes (gadA and gadB) in pathogenic Escherichia coli. Mol Biol Evol 24:2323-33.
6. Boot, I. R., P. Cash, and C. O'Byrne. 2002. Sensing and adapting to acid stress. Antonie Van Leeuwenhoek 81:33-42.
7. Castanie-Cornet, M. P., T. A. Penfound, D. Smith, J. F. Elliott, and J. W. Foster. 1999. Control of acid resistance in Escherichia coli. J Bacteriol 181:3525-35.
8. Christ, D., and J. W. Chin. 2008. Engineering Escherichia coli heat-resistance by synthetic gene amplification. Protein Eng Des Sel 21:121-5.
9. Cupples, C. G., and J. H. Miller. 1988. Effects of amino acid substitutions at the active site in Escherichia coli beta-galactosidase. Genetics 120:637-44.
10. De Champs, C., M. P. Sauvant, C. Chanal, D. Sirot, N. Gazuy, R. Malhuret, J. C. Baguet, and J. Sirot. 1989. Prospective survey of colonization and infection caused by expanded-spectrum-beta-lactamase-producing members of the family Enterobacteriaceae in an intensive care unit. J Clin Microbiol 27:2887-90.
11. de Maagd, R. A., and B. Lugtenberg. 1986. Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components. J Bacteriol 167:1083-5.
12. Edelstone, D. I., and I. R. Holzman. 1981. Oxygen consumption by the gastrointestinal tract and liver in conscious newborn lambs. Am J Physiol 240:G297-304.
13. Foster, J. W. 2004. Escherichia coli acid resistance: tales of an amateur acidophile. Nat Rev Microbiol 2:898-907.
14. Gong, S., H. Richard, and J. W. Foster. 2003. YjdE (AdiC) is the arginine:agmatine antiporter essential for arginine-dependent acid resistance in Escherichia coli. J Bacteriol 185:4402-9.
15. Hong, W., Y. E. Wu, X. Fu, and Z. Chang. Chaperone-dependent mechanisms for acid resistance in enteric bacteria. Trends Microbiol.
16. Hsieh, P. F., H. H. Lin, T. L. Lin, and J. T. Wang. CadC regulates cad and tdc operons in response to gastrointestinal stresses and enhances intestinal colonization of Klebsiella pneumoniae. J Infect Dis 202:52-64.
17. Kashiwagi, K., S. Miyamoto, F. Suzuki, H. Kobayashi, and K. Igarashi. 1992. Excretion of putrescine by the putrescine-ornithine antiporter encoded by the potE gene of Escherichia coli. Proc Natl Acad Sci U S A 89:4529-33.
18. Kashiwagi, K., R. Watanabe, and K. Igarashi. 1994. Involvement of ribonuclease III in the enhancement of expression of the speF-potE operon encoding inducible ornithine decarboxylase and polyamine transport protein. Biochem Biophys Res Commun 200:591-7.
19. Kato, A., H. Ohnishi, K. Yamamoto, E. Furuta, H. Tanabe, and R. Utsumi. 2000. Transcription of emrKY is regulated by the EvgA-EvgS two-component system in Escherichia coli K-12. Biosci Biotechnol Biochem 64:1203-9.
20. Kern, R., A. Malki, J. Abdallah, J. Tagourti, and G. Richarme. 2007. Escherichia coli HdeB is an acid stress chaperone. J Bacteriol 189:603-10.
21. Lin, C. T., T. Y. Huang, W. C. Liang, and H. L. Peng. 2006. Homologous response regulators KvgA, KvhA and KvhR regulate the synthesis of capsular polysaccharide in Klebsiella pneumoniae CG43 in a coordinated manner. J Biochem 140:429-38.
22. Ma, Z., N. Masuda, and J. W. Foster. 2004. Characterization of EvgAS-YdeO-GadE branched regulatory circuit governing glutamate-dependent acid resistance in Escherichia coli. J Bacteriol 186:7378-89.
23. Machuca, A., and A. M. Milagres. 2003. Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus. Lett Appl Microbiol 36:177-81.
24. Malki, A., H. T. Le, S. Milles, R. Kern, T. Caldas, J. Abdallah, and G. Richarme. 2008. Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB. J Biol Chem 283:13679-87.
25. Markowitz, S. M., J. M. Veazey, Jr., F. L. Macrina, C. G. Mayhall, and V. A. Lamb. 1980. Sequential outbreaks of infection due to Klebsiella pneumoniae in a neonatal intensive care unit: implication of a conjugative R plasmid. J Infect Dis 142:106-12.
26. Maroncle, N., C. Rich, and C. Forestier. 2006. The role of Klebsiella pneumoniae urease in intestinal colonization and resistance to gastrointestinal stress. Res Microbiol 157:184-93.
27. Mates, A. K., A. K. Sayed, and J. W. Foster. 2007. Products of the Escherichia coli acid fitness island attenuate metabolite stress at extremely low pH and mediate a cell density-dependent acid resistance. J Bacteriol 189:2759-68.
28. Merdanovic, M., T. Clausen, M. Kaiser, R. Huber, and M. Ehrmann. Protein quality control in the bacterial periplasm. Annu Rev Microbiol 65:149-68.
29. Merrell, D. S., C. Bailey, J. B. Kaper, and A. Camilli. 2001. The ToxR-mediated organic acid tolerance response of Vibrio cholerae requires OmpU. J Bacteriol 183:2746-54.
30. Miethke, M., and M. A. Marahiel. 2007. Siderophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev 71:413-51.
31. Moreau, P. L. 2007. The lysine decarboxylase CadA protects Escherichia coli starved of phosphate against fermentation acids. J Bacteriol 189:2249-61.
32. Nishino, K., and A. Yamaguchi. 2001. Overexpression of the response regulator evgA of the two-component signal transduction system modulates multidrug resistance conferred by multidrug resistance transporters. J Bacteriol 183:1455-8.
33. Podschun, R., and U. Ullmann. 1998. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11:589-603.
34. Rhodius, V. A., W. C. Suh, G. Nonaka, J. West, and C. A. Gross. 2006. Conserved and variable functions of the sigmaE stress response in related genomes. PLoS Biol 4:e2.
35. Rode, T. M., T. Moretro, S. Langsrud, O. Langsrud, G. Vogt, and A. Holck. Responses of Staphylococcus aureus exposed to HCl and organic acid stress. Can J Microbiol 56:777-92.
36. Ron, E. Z. 2009. Editorial: an update on the bacterial stress response. Res Microbiol 160:243-4.
37. Schellhorn, H. E., J. P. Audia, L. I. Wei, and L. Chang. 1998. Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli. J Bacteriol 180:6283-91.
38. Sklar, J. G., T. Wu, D. Kahne, and T. J. Silhavy. 2007. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes Dev 21:2473-84.
39. Skorupski, K., and R. K. Taylor. 1996. Positive selection vectors for allelic exchange. Gene 169:47-52.
40. Smith, J. L. 2003. The role of gastric acid in preventing foodborne disease and how bacteria overcome acid conditions. J Food Prot 66:1292-303.
41. Su, L. H., T. L. Wu, Y. P. Chiu, J. H. Chia, A. J. Kuo, C. F. Sun, T. Y. Lin, and H. S. Leu. 2001. Outbreaks of nosocomial bloodstream infections associated with multiresistant Klebsiella pneumoniae in a pediatric intensive care unit. Chang Gung Med J 24:103-13.
42. Tapley, T. L., T. M. Franzmann, S. Chakraborty, U. Jakob, and J. C. Bardwell. Protein refolding by pH-triggered chaperone binding and release. Proc Natl Acad Sci U S A 107:1071-6.
43. Tramonti, A., M. De Canio, and D. De Biase. 2008. GadX/GadW-dependent regulation of the Escherichia coli acid fitness island: transcriptional control at the gadY-gadW divergent promoters and identification of four novel 42 bp GadX/GadW-specific binding sites. Mol Microbiol 70:965-82.
44. Valderas, M. W., R. B. Alcantara, J. E. Baumgartner, B. H. Bellaire, G. T. Robertson, W. L. Ng, J. M. Richardson, M. E. Winkler, and R. M. Roop, 2nd. 2005. Role of HdeA in acid resistance and virulence in Brucella abortus 2308. Vet Microbiol 107:307-12.
45. Wang, W., T. Rasmussen, A. J. Harding, N. A. Booth, I. R. Booth, and J. H. Naismith. Salt bridges regulate both dimer formation and monomeric flexibility in HdeB and may have a role in periplasmic chaperone function. J Mol Biol 415:538-46.
46. Watson, D., R. D. Sleator, C. Hill, and C. G. Gahan. 2008. Enhancing bile tolerance improves survival and persistence of Bifidobacterium and Lactococcus in the murine gastrointestinal tract. BMC Microbiol 8:176.
47. Wollmann, P., and K. Zeth. 2007. The structure of RseB: a sensor in periplasmic stress response of E. coli. J Mol Biol 372:927-41.
48. Yong, D., M. A. Toleman, C. G. Giske, H. S. Cho, K. Sundman, K. Lee, and T. R. Walsh. 2009. Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 53:5046-54.
49. Zhang, M., S. Lin, X. Song, J. Liu, Y. Fu, X. Ge, X. Fu, Z. Chang, and P. R. Chen. A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance. Nat Chem Biol 7:671-7.
50. Zhao, B., and W. A. Houry. Acid stress response in enteropathogenic gammaproteobacteria: an aptitude for survival. Biochem Cell Biol 88:301-14.