以基因微陣列比較RNA表現發現台灣引起肝膿瘍之克雷伯氏肺炎桿菌與allantoin代謝有關之特有的染色體片段

Isolation of a Chromosomal Region Associated with Allantoin Metabolism and Liver Infection in Klebsiella pneumoniae by Microarray

10.6342/NTU.2004.00155

周慧琦

肝感染 ； 克雷伯氏肺炎桿菌 ； 微陣列 ； 肝膿瘍 ； 致病因子 ； microarray ； allantoin ； virulence factor ； liver abscess ； Klebsiella pneumoniae

臺灣大學微生物學研究所學位論文

2004年

博士

王錦堂

繁體中文

克雷伯氏肺炎桿菌(Klebsiella pneumoniae)所引發的原發性肝膿瘍 (primary liver abscess)，目前已成為台灣成人常見的社區感染症之一，然而其致病機轉尚未完全明瞭。因此，我們利用基因微陣列 (microarray)來尋找可能存在於引起肝感染克雷伯氏肺炎桿菌 (liver infection K. pneumoniae) 特有的致病因子(virulence factor)。比較引起肝感染與不引起肝感染 (non-liver infection) 克雷伯氏肺炎桿菌RNA表現的差異，發現於肝感染克雷伯氏肺炎桿菌表現值大於非肝感染克雷伯氏肺炎桿菌表現值兩倍以上者有14個殖株 (clones)。其中3個殖株(clone-1, 2, 6)為相同，並且5’ 端有1374 bp的DNA序列無法在克雷伯氏肺炎桿菌MGH 78578找到相對應的位置。菌株MGH 78578為非肝感染菌株，其基因體序列解碼目前正由美國華盛頓大學基因體定序中心 (http//genome.wustl.edu/gsc) 進行中。 DNA定序的結果顯示MGH 78578染色體上一段149 bp的片段在肝感染克雷伯氏肺炎桿菌NTUH-K2044被一段總長21745 bp的片段所取代。此21745 bp片段總共包括19個開放讀架 (open reading frame)，其中14個開放讀架證明和allantoin代謝相關。另一方面，這個片段於肝感染及非肝感染克雷伯氏肺炎桿菌的分佈情形有極顯著的差異 (19/32比2/94, p=0.0001, x2 test)。同時，在動物實驗也發現，同樣以胃內感染方式注射104 CFU時，NTUH-K2044在老鼠能造成嚴重的肝膿瘍及致死，而在NTUH-K2044染色體上將具有正向調控allantoin operons弁鄋滌穧]allS作基因剔除 (knock out) 所得的缺陷株 K2044(

Klebsiella pneumoniae liver abscess with metastatic complications is an emerging infectious disease in Taiwan. To identify genes associated with liver infection, we used a DNA microarray to compare the transcriptional profiles between three strains causing liver abscess and three strains without liver infection. There were 14 clones that revealed higher RNA expression levels in the three liver infection strains and three of these 14 clones contained a region that was absent in MGH 78578. The strain of K. pneumoniae MGH 78578 was isolated from a patient without liver abscess and was chosen for full genome sequencing by the Genomic Sequencing Center at Washington University (St. Louis, MO) (http//genome.wustl.edu/gsc). Sequencing of the clones revealed the replacement of 149 bp of MGH 78578 with a 21,745 bp fragment in a liver infection strain, NTUH-K2044. This 21,745-bp fragment contained 19 open reading frames, 14 of which were proven to be associated with allantoin metabolism. The K2044(

1. 4.Ang, S., C. Z. Lee, K. Peck, M. Sindici, U. Matrubutham, M. A. Gleeson, and J. T. Wang. 2001. Acid-induced gene expression in Helicobacter pylori: study in genomic scale by microarray. Infect. Immun. 69:1679-1686.
   連結：
2. 5.Bartlett, J. g., P. O’keefe, F. P. Tally, T. J. Louie, and S. L. Gorbach. 1986. Bacteriology of hospital-acquired pneumonia. Arch. Intern. Med. 146:868-871.
   連結：
3. 6.Benzie, I. F. F., Chung, W. Y., and B. Tomlinson. 1999. Simultaneous measurement of allantoin and urate in plasma: analytical evaluation and potential clinical application in oxidant:antioxidant balance studies. Clin. Chem. 45:901-904.
   連結：
4. 7.Boronat, A., and J. Aguilar. 1979. Rhamnose-induced propanediol oxidoreductase in Escherichia coli: purification, properities, and comparison with the fucose-induced enzyme. J. Bacteriol. 140:320-326.
   連結：
5. 8.Carpenter, J. L. 1990. Klebsiella pulmonary infections: occurrence at one medical center and review. Rev. Infect. Dis. 12:672-682.
   連結：
6. 9.Chang, H. Y., J. H. Lee, W. L. Deng, T. F. Fu, and H. L. Peng. 1996. Virulence and outer membrane properties of a galU mutant of Klebsiella pneumoniae CG43. Microb. Pathog. 20:255-261.
   連結：
7. 10.Chang, S. C., C. T. Fang, P. R. Hsueh, Y. C. Chen, and K. T. Luh. 2000. Klebsiella pneumoniae isolates causing liver abscess in Taiwan. Diagn. Microbiol. Infect. Dis. 37: 279–284.
   連結：
8. 11.Chen, J. W., R. Wu, P. C. Yang, J. Y. Huang, Y. P. Sher, M. H. Han, W. C. Kao, P. J. Lee, T. F. Chiu, F. Chang, et al. 1998. Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection. Genomics 51:313-324.
   連結：
9. 13.Cheng, D. L., Y. C. Liu, M. Y. Yen, C. Y. Liu, and R. S. Wang. 1991. Septic metastatic lesions of pyogenic liver abscess. Their association with Klebsiella pneumoniae bacteremia in diabetic patients. Arch. Intern. Med. 151: 1557– 1559.
   連結：
10. 14.Cheng, H. P., L. K. Siu, and F. Y. Chang. 2003. Extended-spectrum cephalosporin compared to cefazolin for treatment of Klebsiella pneumoniae-caused liver abscess. Antimicrob. Agents Chemother. 47:2088-2092.
    連結：
11. 15.Cherubin, C. E., and R. H. K. Eng. 1986. Experience with the use of cefotaxime in the treatment of bacterial meningitis. Am. J. Med. 80:398-404.
    連結：
12. 16.Chiu, C. T., D. Y. Lin, and Y. F. Liaw. 1988. Metastatic septic endophthalmitis in pyogenic liver abscess. J. Clin. Gastroenterol. 10: 524–527.
    連結：
13. 19.Di Figlia, S. E., and C. Cramer. 1951. Friedlander’s bacillus meningitis in a case with liver abscess and recurrent bacteremia and analysis of cases receiving specific therapy. N. Y. State J. Med. 15:761-765.
    連結：
14. 21.de la Torre, M. G., J. romero-Vivas, J. Martinez-Beltran, A. Guerrero, M. Meseguer, and E. Bouza. 1985. Klebsiella bacteremia: an analysis of 100 episodes. Rev. Infect. Dis. 7:143-150.
    連結：
15. 23.Domenico, P., W. G. Johanson, and D. C. Straus. 1982. Lobar pneumonia in rats produced by clinical isolates of Klebsiella pneumoniae. Infect. Immun. 37:327-335.
    連結：
16. 26.Fang, C. T., Y. P. Chuang, C. T. Sun, S. C. Chang, and J. T. Wang. 2004. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J. Exp. Med. 199:697-705.
    連結：
17. 27.Fang, C. T., H. C. Chen, Y. P. Chuang, S. C. Chang, and J. T. Wang. 2002. Cloning of a cation efflux pump gene associated with chlorhexidine resistance in Klebsiella pneumoniae. Antimicrob. Agents Chemother. 46:2024-2028.
    連結：
18. 28.Fang, C. T., Y. C. Chen, S. C. Chang, W. Y. Sau, and K. T. Luh. 2000. Klebsiella pneumoniae meningitis: timing of antimicrobial therapy and prognosis. QJM. 93:45-53.
    連結：
19. 30.Frees, D., S. N. A. Qazi, P. J. Hill, and H. Ingmer. 2003. Alternative roles of ClpX and ClpP in Staphylococcus aureus stress tolerance and virulence. Mol. Microbiol. 48:1565-1578.
    連結：
20. 31.Fung, C. P., F. Y. Chang, S. C. Lee, B. S. Hu, B. I. Kuo, C. Y. Liu, M. Ho, and L. K. Siu. 2002. A global emerging disease of Klebsiella pneumoniae liver abscess: is serotype K1 an important factor for complicated endophthalmitis? Gut. 50: 420–424.
    連結：
21. 32.Gaillot, O., E. Pellegrini, S. Bregenholt, S. Nair, and P. Berche. 2000. The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol. Microbiol. 35:1286-1294.
    連結：
22. 33.Garaizar, J., S. Porwollik, A. Echeita, A. Rementeria, S. Herrera, RM. Wong, J. Frye, MA. Usera, and M. McClelland. 2002. DNA microarray-based typing of an atypical monophasic Salmonella enterica serovar. J. Clin. Microbiol. 40:2074-2078.
    連結：
23. 34.Gort, A. S., and V. L. Miller. 2000. Identification and characterization of Yersinia enterocolitica genes induced during systemic infection. Infect. Immun. 68: 6633-6642.
    連結：
24. 35.Grootveld, M., and B. Hallwell. 1987. Measurement of allantoin and uric acid in human body fluids: a potential index of free-radical ractions in vivo? Biochem. J. 243:803-808.
    連結：
25. 36.Hacker, J., and J. B. Kaper. 2000. Pathogenicity islands and the evolution of microbes. Annu. Rev. Microbiol. 54:641-679.
    連結：
26. 37.Herrero M., V. Lorenzo, and K. N. Timmis. 1990. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J. Bacteriol. 172:6557-6567.
    連結：
27. 38.Highsmith, A. K., and W. R. Jarvis. 1985. Klebsiella pneumoniae: selected virulence factors that contribute to pathogenicity. Infect. Control 6:75-77
    連結：
28. 39.Jong G. M., T. R. Hsiue, C. R. Chen, H. Y. Chang, C. W. Chen. 1995. Rapidly fatal outcome of bacteremic Klebsiella pneumoniae pneumonia in alclholics. Chest 107:214-217.
    連結：
29. 40.Kastenbauer, S., Y. Koedel, B. F. Becker, and H. W. Pfister. 2002. Oxidative stree in bacterial meningitis in humans. Neurology. 58:186-191.
    連結：
30. 41.Ko, W. C., D. L. Paterson, A. J. Sagnimeni, D. S. Hansen, A. Von Gottberg, S. Mohapatra, J. M. Casellas, H. Goossens, L. Mulazimoglu, G. Trenholme, et al. 2002. Community-acquired Klebsiella pneumoniae bacteremia: global differences in clinical patterns. Emerg. Infect. Dis. 8: 160–166.
    連結：
31. 42.Kobayashi, N., K. Nishino, and A. Yamaguchi. 2001. Novel macrolid-specific ABC-type efflux transporter in Escherichia coli. J. Bacteriol. 183:5639-5644.
    連結：
32. 43.Kruger, E., E. Witt, S. Ohlmeier, R. Hanschke, and M. Hecker. 2000. The clp proteases of Bacillus subtilis are directly involved in degradation of misfolded proteins. J. Bacteriol. 182:3259-3265.
    連結：
33. 44.Liu, Y. C., D. L. Cheng, and C. L. Lin. 1986. Klebsiella pneumoniae liver abscess associated with septic endophthalmitis. Arch. Intern. Med. 146: 1913–1916.
    連結：
34. 45.Marzluf, G. A. 1997. Genetic regulation of nitrogen metabolism in the fungi. Microbiol. Mol. Biol. Rev. 61:17-32.
    連結：
35. 48.Moison, R., A. de Beaufort, A. Hassnoot, T. Dubbelman, D. van-Zoeren-Grubben, and H. Berger. 1997. Uric acid and ascorbic acid redox ratios in plasma and tracheal aspirate of preterm babies with acute and chronic lung disease. Free Radic. Biol. Med. 23:226-234.
    連結：
36. 49.Montgomerie, J. Z. 1979. Epidemiology of Klebsiella and hospital-associated infections. Rev. Infect. Dis. 1:736-753
    連結：
37. 50.Moore, T. D. E., and P. F. Sparling. 1995. Isolation and identification of a glutathione peroxidase homolog gene, gpxA, present in Neisseria meningitides but absent in Neisseria gonorroeae. 63:1603-1607.
    連結：
38. 51.Ogihara, T., H. Kim, K. Hirano, M. Imanishi, H. Ogihara, H. Tamai, R. Okamoto, and M. Mino. 1998. Oxidation products of uric acid and ascorbic acid in preterm infants with chronic lung disease. Biol. Neonate. 73:24-33.
    連結：
39. 52.Old, D. C., A. Tavendale, and B. W. Senior. 1985. A comparative study of the type-3 fimbriae of Klebsiella species. J. Med. Microbiol. 20:203-214
    連結：
40. 54.Ørskov, I., and M. A. Fife-Asbury. 1977. New Klebsiella capsular antigen, K82, and the deletion of five of those previously assigned. Int. J. Syst. Bacteriol. 27:386-387.
    連結：
41. 55.Podschun, R., P. Heineken, and H. G. Sonntag. 1987. Hemagglutinins and adherence properties to HeLa and Intestine 407 cells of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Zentbl. Bakteriol. Mikrobiol. Hyg. Ser. A 263:585-593.
    連結：
42. 57.Pederson, K. J., S. Carlson, and D. E. Pierson. 1997. The ClpP protein, a subunit of the Clp protease, modulates ail gene expression in Yersinia enterocolitica. Mol. Microbiol. 26:99-107.
    連結：
43. 58.Pomposiello, P. J., and B. Demple. 2000. Identification of SoxS-regulated genes in Salmonella enterica serovar Typhimurium. J. Bacteriol. 182:23-29.
    連結：
44. 61.Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27: 493–497.
    連結：
45. 62.Rintoul M. R., E. Cusa, L. Baldoma, J. Badia, L. Reitzer, and J. Aguilar. 2002. Regulation of the Escherichia coli allantoin regulon: coordinated function of the repressor AllR and the activator AllS. J. Mol. Biol. 324:599-610.
    連結：
46. 64.Sakai T., K. Kanai, K. Osatomi, and K. Yoshikoshi. 2003. Identification of a 19.3-kDa protein in MRHA-positive Edwardsiella tarda: putative fimbrial major subunit. FEMS Microbiol. Lett. 226:127-33.
    連結：
47. 66.Seeto, R. K., and D. C. Rocky. 1996. Pyogenic liver abscess: change in etiology, management, and outcome. Medicine. 75:99-113.
    連結：
48. 69.Wang, F. D., L. S. Wang, Y. C. Liu, C. L. Lin, and W. W. Wong. 1989. Successful treatment of metastatic endophthalmitis. Ophthalmologica. 198:124-128.
    連結：
49. 70.Wang, J. H., Y. C. Liu, S. S. Lee, M. Y. Yen, Y. S. Chen, J. H. Wang, S. R. Wang, and H. H. Lin. 1998. Primary liver abscess due to Klebsiella pneumoniae in Taiwan. Clin. Infect. Dis. 26: 1434–1438.
    連結：
50. 71.Wang, L. S., F. Y. Lee, D. L. Cheng, C. Y. Liu, D. R. Hinthorn, and P. M. Jost. Klebsiella pneumoniae bacteremia: a analysis of 100 episodes. Rev. Infect. Dis. 7:143-150.
    連結：
51. 72.Williams, P., P. A. Lambert, M. R. W. Brown, and R. J. Jones. 1983. The role of the O and K antigens in determining the resistance of Klebsiella aerogenes to serum killing and phagocytosis. J. Gen. Microbiol. 129:2181-2191.
    連結：
52. 73.Yanagawa, T., H. Nakamura, I. Takei, H. Maruyama, K. Kataoka, T. Saruta, and Y. Kobayashi. 1989. Klebsiella pneumoniae meningitis associated with liver abscess: a case report. Jpn. J. antibiot. 42:2135-2140.
    連結：
53. 74.Yeh, Y. C., K. C. Chang, C. T. Fang, J. C. Yang, and J. T. Wang. 2002. Association of metronidazole resistance and natural competence in Helicobacter pylor. Antimicrob. Agents Chemother. 46(5):1564-7.
    連結：
54. 75.Yeoh, K. G., I. Yap, S. T. Wong, A. Wee, R. Guan, and J. Y. Kang. 1997. Tropical liver abscess. Postgrad. Med. J. 73:89-92.
    連結：
55. 1.Abbott, S. 1999. Klebsiella, Enterobacter, Citrobacter and Serratia. p. 475–482. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
56. 2.Alberti, S., D. Alvarez, S. Merino, M. T. Casado, F. Vivanco, J. M. Tomas, and V. J. Benedi. 1996. Analysis of complement C3 deposition and degradation on Klebsiella pneumoniae. Infect. Immun. 64:4726-4732.
57. 3.Alberti, S., G. Marques, S. Camprubi, S. Merino, J. M. Tomas, F. Vivanco, and V. J. Benedi. 1993. C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins. Infect. Immun. 61:852-860.
58. 12.Cheng, D. L., Y. C. Liu, M. Y. Yen, C. Y. Liu, F. W. Shi and L. S. Wang. 1990. Pyogenic liver abscess: clinical manifestations and value of percutaneous catheter drainage treatment. J. Formos. Med. Assoc. 89: 571-576.
59. 17.Cryz, S. J., E. Furer, and R. Germanier. 1984. Experimental Klebsiella pneumoniae burn wound sepsis: role of capsular polysaccharide. Infect. Immun. 43:440-441.
60. 18.Cusa, E., N. Obradors, L. Baldoma, J. Badia, and J. Aguilar. 1999. Genetic analysis of a chromosomal region containing genes required for assimilation of allantoin nitrogen and linked glyoxylate metabolism in Escherichia coli. J. Bacteriol. 181:7479-7484.
61. 20.Darfeuille-Michaud, A., C. Jallat, D. Aubel, D. Sirot, C. Rich, J. Sirot, and B. Joly. 1992. R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections. Infect. Immun. 60:44-45.
62. 22.Di Martino, P., V. Livrelli, D. Sirot, B. Joly, and A. Darfeuille-Michaud. 1996. A new fimbrial antigen harbored by CAZ-5/SHV-4-producing Klebsiella pneumoniae strains involved in nosocomial infections. Infect. Immun. 64:2266-2273.
63. 24.Ehrenwort, L., and H. Baer. 1956. The pathogenicity of Klebsiella pneumoniae for mice: the relationship to the quantity and rate of production of type-specific capsular polysaccharide. J. Bacteriol. 72:713-717.
64. 25.Eisenstein, B. I., and D. F. Zaleznik. 2000. Enterobacteriaceae. p.2294-2309. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases, 5th ed. Philadelphia: Chorchill-Livingstone.
65. 29.Favre-Bonte, S., A. Darfeuille-Michaud, and C. Forestier. 1995. Aggregative adherence of Klebsiella pneumoniae to human Intestine-407 cells. Infect. Immun. 63:1318-1328
66. 46.McCallum, K. L., G. Schoenhals, D. Laakso, B. Clarke, and C. Whitfield. 1989. A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing. Infect. Immun. 57:3816-3822
67. 47.Merino, S., S. Camprubí, S. Albertí, V. J. Benedí, and J. M. Tomás. 1992. Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing. Infect. Immun. 60:2529-2535
68. 53.Old, R. W., and S. B. Primrose. 1996. Principles of gene manipulation. 5th ed. Oxford: Blackwell Science.
69. 56.Podschun, R., and U. Ullmann. 1998. Klebsiella spp. As nosocomial pathogens:epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microb. Rev. 11:589-603.
70. 59.Przondo-Hessek, A., and G. Pulverer. 1983. Hemagglutinins of Klebsiella pneumoniae and Klebsiella oxytoca. Zentbl. Bakteriol. Mikrobiol. Hyg. Ser. A 255:472-478.
71. 60.Reddy, K. J. and M. Gilman. 1987. Preparation and analysis of RNA, p.4.4.1-4.4.7. In Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology, vol. 1. John Wiley & Sons. Inc, USA.
72. 63.Rouf, M. A., and R. f. Lomprey. 1968. Degradation of uric acid by certain aerobic bacteria. J. Bacteriol. 96: 617-622.
73. 65.Salky, B. A., A. Kaynon, J. J. Bauer, I. M. Gelernt, and I. Kreel. 1982. Ruptured hepatic abscess: a rare cause of spontaneous pneumoperitoneum. Am. J. Gastroenterol. 77:880-881.
74. 67.Straus, D. C., D. L. Atkisson, and C. W. Garner. 1985. Importance of a lipopolysaccharide-containing extracellular toxic complex in infections produced by Klebsiella pneumoniae. Infect. Immun. 50:787-795.
75. 68.Tomás, J. M., V. J. Benedí, B. Ciurana, and J. Jofre. 1986. Role of capsule and O antigen in resistance of Klebsiella pneumoniae to serum bactericidal activity. Infect. Immun. 54:85-89.
76. 76.鄭德齡，劉永慶. 1997. Klebsiella pneumoniae 肝膿瘍：臺灣地區的特產. J. Infect. Dis. Soc. ROC 1:1-3.

1. 鐘純珊(2009)。克雷白氏肺炎桿菌NTUH-K2044 中纖毛黏附蛋白KpfD的功能性分析。交通大學分子醫學與生物工程研究所學位論文。2009。1-54。