摘要
从唾液和龈沟液中持续分泌的尿素被口腔细菌尿素酶快速分解产生氨和二氧化碳。尿素水解的碱性产物通过中和细菌产酸以维持牙菌斑的中性pH值环境，有助于稳定牙菌斑菌群平衡，防止牙釉质脱矿和促进牙釉质再矿化。因此，尿素水解被认为对防止龋病发生起到了重要作用。迄今为止，关于牙菌斑中哪些细菌具有尿素酶活性以及哪些细菌决定牙菌斑尿素酶活性的问题尚不清楚，对口腔细菌尿素酶基因表达的调控机制缺乏认识。本研究首先对牙菌斑中常驻细菌进行尿素酶活性检测，比较细菌间尿素酶的酶促动力学特征，寻找决定牙菌斑尿素酶活性的关键细菌。在此基础上，以内氏放线菌作为研究对象，了解细菌尿素代谢的生物学特征，模拟内氏放线菌在牙菌斑中的生物膜状态，探索环境因素和生存状态对细菌尿素酶活性和尿素酶基因表达的影响。
方法 采用连续培养的恒化器模型培养内氏放线菌单菌种生物膜，使用酶底物反应法和Real-time PCR技术分别在生化水平和基因水平检测氮源物质、糖源物质、pH值和清除率对尿素酶活性和尿素酶UreC基因表达的影响，同时比较细菌在膜状态下和浮游状态下尿素酶活性和尿素酶UreC基因表达的差异。
结果 研究发现，牙菌斑生物膜中细菌有活跃的尿素代谢活动，唾液链球菌，内氏放线菌能稳定表达尿素酶活性，血液链球菌和粘性放线菌尿素
Urea are secreted from saliva and gingival crevicular fluid continuously and rapidly broken down by urease enzymes of oral microflora to ammonia and carbonhydrate. Alkali production from ureolysis could neutralize acid end products to stabilize pH homotasis in dental biofilms, so be helpful to stabilize microflora balance, inhibit enamel demineralization and promote mineral deposition. Thus, ureolysis is considered to be an important factor to prevent dental caries. High level of specific urease activity was detected in dental plaque, but understanding of the molecular characteristics of the urease enzymes of oral bacteria and the critical role that ureolysis play in ecological balance of dental biofilms are very limited. In this study, firsty, we concentrated on the examination of ureolytic bacteria in dental biofilms and compare the biochemical characteristics of the enzyme from different strains. Based on the result of the first part, we took Actinomyces neaslundii as research objective to study the physiological characteristics of ureolysis and effects of envirometal factors and growth state on the urease expression. Methods Continuous culture chemostat system was used to establish a nono-specie biofilms of A. neaslundii to study the effect of enviromental factors on the urease gene expression. In this part, biochemical methods and Real-time PCR techniques were used to detect the urease specific activity and urease mRNA levels as a function of pH, dilution rate, carbohydrate and nitrogen availability in fluid phase of culture medium. At the same time, the
引文
1. Buck GE. Campylobacter pylori and gastroduodenal disease. Clin Microbiol Rev. 1990; 3(1): 1-12
2. Musher DM, Griffith DP, Yawn D, et al. Role of urease in pyelonephritis resulting from urinary tract infection with Proteus. J. Infect.Dis. 1975; 131(2): 177-181
3. McKnight SL, Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science.1982; 217(4557):316-324
4. Golub LM, Borden SM, Kleinberg I. Urea content of gingival crevicular fluid and its relation to periodontal diseases in humans. J. Periodont. Res. 1971; 6(4): 243-251
5. Sissons CH, Cutress TW, Pearce EI. Kinetics and product stoichiometry of ureolysis by human salivary bacteria and artificial mouth plaques. Arch. Oral Biol. 1985; 30(11-12):781-790
6. Margolis HC, Duckworth JH, Moreno EC. Composition of pooled resting plaque fluid from caries-free and caries susceptible individuals. J. Dent. Res. 1988; 67(12): 1468-1475
7. Peterson S, Woodhead J, Crall J. Caries resistance in children with chronic renal failure: plaque pH, salivary pH, and salivary composition. Pediatr. Res. 1985; 19(8):796-799
8. Burne RA. Oral streptococci... products of their environment. J. Dent. Res. 1998; 77(3): 445-452
9. Bowden, GHW, Ellwood DC, Hamilton IR. 1979. Microbial ecology of the oral cavity, p. 135-217. In M. Alexander (ed.), Advances in microbial ecology. Plenum Press, New York, NY
10. Bradshaw DJ, Mckee AS, Marsh PD. Effects of carbohydrate pulses and pH on population shifts within oral microbial communities in vitro. J. Dent. Res. 1989; 68(9):1298-1302
11. Pearce EI, Schamschula RG, Cooper MH. Increases in fluoride, calcium, and phosphate in dental plaque resulting from the use of a mineralizing mouthrinse containing urea and monofluorophosphate. J. Dent. Res. 1983; 62(7): 818-820
12. Pearce EI, Wakefield JS, Sissons CH. Therapeutic mineral enrichment of dental plaque visualized by transmission electron microscopy. J. Dent. Res. 1991; 70(2):90-94
13. Epstein SR, Mandel I, Scopp IW. Salivary composition and calculus formation in patients undergoing hemodialysis. J. Peridontol. 1980; 51(6):336-338
14. Mandel ID, Thompson RH. The chemistry of paratid and submaxillary saliva in heavy calculus formers and non-formers. J. Peridontol. 1967; 38(4): 310-315
15. Helegland K. pH and the effect of NH4Cl on human gingival fibroblasts. Scand J. Dent. Res. 1985; 93(1): 39-45
16. Hattab F, Frostell G. The release of fluoride from two products of alginate impression materials. Acta Odontol. Scand. 1980; 38(3): 385-935
17. Gallagher IH, Pearce EIF, Cutress TW. The ureolytic microflora of immature dental plaque before and after rinsing with a urea-based mineralizing solution. J.Dent Res. 1984; 63,(8): 1037-1039
18. Salako NO, Kleinberg I. Incidence of selected ureolytic bacteria in human dental plaque from sites with differing salivary access. Arch Oral Biol. 1989;34(10):787-91.
19. Cook AR. A chemically-defined medium for the growth of a ureolytic strain of Streptococcus faecium. J.gen.microbial. 1976; 92(2): 49-58
20. Wozny MA, Bryant MP, Holdemann LV, et al. Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces.Appl.envion.Microbiol. 1977; 33(5): 1097-1104
21.Frostell G The effect of chewing on the ph of dental plaques after carbohydrate consumption. Acta Odontol. Scand. 1974; 32(2): 79-82
22. Mobley HL, Hausinger RP. Microbiol ureases: significance, regulation, and molecular characterization. Microbiol. Rev. 1989; 53(1):85-108.
23. Collins CM, Dorazio SEF. Bacterial ureases: structure, regulation of expression and role in pathogenesis. Mol. Microbiol. 1993; 9(5):907-913
24. Mobley HL, Island MD, Hausinger RP. Molecular biology of ureases. Microbiol. Rev. 1995; 59(3):451 -480
25. Jabri E, Carr MB, Hausinger RP, et al. Science. 1995; 268(5213): 998-1004
26. Lee MH, Mulrooney SB, Rener MJ, et al. Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J. Bacteriol. 1992; 174(13): 4324-4330
27. Mulrooney SB, Hausinger RP. Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation. J. Bacteriol. 1990; 172(10): 5837-5843
28. Maeda M, Hindaka M, Nakamura A, et al. Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli. J. Bacteriol. 1994; 176(2): 3432-442
29. Mobley HL, Garner RM, Bauerfeld P. Helicobacter pylori nickel-transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions. Mol.Microbiol. 1995; 16(1): 97-109
30. Chen YY, Weaver CA, Mendelsohn DR, et al. Transcriptional regulation of the Streptococcus salivarius 57.I urease operon. J Bacteriol. 1998; 180(21): 5769-5775.
31. Weeks DL, Eskandari S, Scott DR, et al. A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science. 2000; 287(5452): 482-485.
32. Dorazio SEF, Collins CM. The plasmid-encoded urease gene cluster of the family Enterobacteriaceae is positively regulated by UreR, a member of the AraC family of transcriptional activators. J. Bacteriol. 1993; 175(11): 3459-3467
33. Nicholson EB, Concaugh EA, Foxall PA, et al. Proteus mirabilis urease: transcriptional regulation by UreR. J. Bacteriol. 1993; 175(2): 465-473
34. Sissons CH, Hancock EM. Cutress TW. The source of variation in ureolysis in artificial plaques cultured from human salivary bacteria. Archs Oral Biol. 1988; 33(10): 721-726
35. Sissons CH, Loong PC, Hancock EM. Electrophoretic analysis of ureases in Streptococcus salivarius and in saliva. Oral Microbiol Immunol. 1989; 4(4): 211-218
36. Kopstein J, Wrong OM. The origin and fate of salivary urea and ammonia in man. 1977, Clin. Sci. Molec. Med.52(1): 9-17
37. Sissons CH, Hancock EM. Urease activity in Streptococcus salivarius at low pH. Archs Oral Biol. 1993; 38(6): 507-516
38. Hillman JD, Chen A, Snoep JL. Genetic and physiological analysis of the lethal effect of L-(+)-lactate dehydrogenase deficiency in Streptococcus mutans: complementation by alcohol dehydrogenase from Zymomonas mobilis. Infect. Immun. 1996; 64(10):4319-4323
39. Kubo S, Kubota H, Ohnishi Y, et al. Expression and secretion of an Arthrobacter dextranase in the oral bacterium Streptococcus gordonii. Infect. Immun. 1993; 61(10): 4375-4381
40. Clancy KA, Burne RA. Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity. FEMS Microbiol. Lett. 1997; 151(2):205-211
41. Morou-Bermudez E, Burne RA. Analysis of Urease Expression in Actinomyces naeslundii WVU45. Infect Immun, 2000; 68(12): 6670-6676
42. Chen YY, Clancy KA, Burne RA. Streptococcus salivarius Urease: Genetic and Biochemical Characterization and Expression in a Dental Plaque Streptococcus. Infect Immun, 1996; 64(2): 585-592
43. Bowden GH, Hardie JM, Fillery ED. Antigens from Actinomyces species and their value in identification. J. Dent. Res, 1976; 55: A192-A204
44. Kleiner D. Energy expenditure for cyclic retention of NH3/NH4+ during N2 fixation by Klebsiella pneumoniae. FEBS Lett. 1985; 187(2): 237-239
45. Chen YY, Weaver CA, Burne RA. Dual functions of Streptococcus salivarius urease. J Bacteriol, 2000; 182(16): 4667-4669
46. Morou-Bermudez E, Burne RA. Genetic and physiologic characterization of urease of Actinomyces naeslundii. Infect. Immun, 1999,67(2): 504-512
47. Bradshaw DJ, Mckee AS, Marsh PD. Effects of carbohydrate pulses and pH on population shifts within oral microbial communities in vitro. J Dent Res. 1989, 68(9): 1298-13027.
48. Clancy KA, Burne RA. Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity. FEMS Microbiol. Lett, 1997,151(2): 205-211
49. Chen YY, Burne RA. Analysis of Streptococcus salivarius urease expression using continuous chemostat culture. FEMS Microbiol. Lett, 1996,135(2-3): 223-229
50. Li YH, Chen YM, Burne RA. Regulation of urease gene expression by Streptococcus salivarius growing in biofilms. Environ Microbiol 2000, 2(2): 169-177
51. Burne RA, Chen YM. The use of continuous flow bioreactors to explore gene expression and physiology of suspended and adherent populations of oral streptococci. Methods Cell Sci. 1998; 20(2):181-190
52. Li YH, Burne RA. Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate. Microbiology. 2000; 147(Pt 10):2841-2848
53. Bowden GH, Hamilton IR.Survival of oral bacteria. Crit Rev Oral Biol Med. 1998; 9(1): 54-85.
54. Sissons CH, Hancock EM, Perinpanayagam HE, et al. The bacteria responsible for ureolysis in artificial dental plaque. Arch Oral Biol. 1988; 33(10): 727-733.
1. Buck GE. Campylobacter pylori and gastroduodenal disease. Clin Microbiol Rev. 1990; 3(1): 1-12
2. Musher DM, Griffith DP, Yawn D, et al. Role of urease in pyelonephritis resulting from urinary tract infection with Proteus. J. Infect.Dis. 1975; 131(2): 177-181
3. McKnight SL, Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science.1982; 217(4557):316-324
4. Golub LM, Borden SM, Kleinberg I. Urea content of gingival crevicular fluid and its relation to periodontal diseases in humans. J. Periodont. Res. 1971; 6(4): 243-251
5. Sissons CH, Cutress TW, Pearce EI. Kinetics and product stoichiometry of ureolysis by human salivary bacteria and artificial mouth plaques. Arch. Oral Biol. 1985; 30(11-12):781-790
6. Margolis HC, Duckworth JH, Moreno EC. Composition of pooled resting plaque fluid from caries-free and caries susceptible individuals. J. Dent. Res. 1988; 67(12): 1468-1475
7. Peterson S, Woodhead J, Crall J. Caries resistance in children with chronic renal failure: plaque pH, salivary pH, and salivary composition. Pediatr. Res. 1985; 19(8):796-799
8. Burne RA. Oral streptococci... products of their environment. J. Dent. Res. 1998; 77(3): 445-452
9. Bowden, GHW, Ellwood DC, Hamilton IR. 1979. Microbial ecology of the oral cavity, p. 135-217. In M. Alexander (ed.), Advances in microbial ecology. Plenum Press, New York, NY
10. Bradshaw DJ, Mckee AS, Marsh PD. Effects of carbohydrate pulses and pH on population shifts within oral microbial communities in vitro. J. Dent. Res. 1989; 68(9):1298-1302
11. Pearce EI, Schamschula RG, Cooper MH. Increases in fluoride, calcium, and phosphate in dental plaque resulting from the use of a mineralizing mouthrinse containing urea and monofluorophosphate. J. Dent. Res. 1983; 62(7): 818-820
12. Pearce EI, Wakefield JS, Sissons CH. Therapeutic mineral enrichment of dental plaque visualized by transmission electron microscopy. J. Dent. Res. 1991;70(2):90-94
13. Epstein SR, Mandel I, Scopp IW. Salivary composition and calculus formation in patients undergoing hemodialysis. J. Peridontol. 1980; 51(6):336-338
14. Mandel ID, Thompson RH. The chemistry of paratid and submaxillary saliva in heavy calculus formers and non-formers. J. Peridontol. 1967; 38(4): 310-315
15. Helegland K. pH and the effect of NH4C1 on human gingival fibroblasts. Scand J. Dent. Res. 1985; 93(1): 39-45
16. Hattab F, Frostell G. The release of fluoride from two products of alginate impression materials. Acta Odontol. Scand. 1980; 38(3): 385-935
17. Gallagher IH, Pearce EIF, Cutress TW. The ureolytic microflora of immature dental plaque before and after rinsing with a urea-based mineralizing solution. J.Dent Res. 1984; 63,(8): 1037-1039
18. Salako NO, Kleinberg I. Incidence of selected ureolytic bacteria in human dental plaque from sites with differing salivary access. Arch Oral Biol. 1989;34(10):787-91.
19. Cook AR. A chemically-defined medium for the growth of a ureolytic strain of Streptococcus faecium. J.gen.microbial. 1976; 92(2): 49-58
20. Wozny MA, Bryant MP, Holdemann LV, et al. Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces. Appl.envion.Microbiol. 1977; 33(5): 1097-1104
21.Frostell G. The effect of chewing on the ph of dental plaques after carbohydrate consumption. Acta Odontol. Scand. 1974; 32(2): 79-82
22. Mobley HL, Hausinger RP. Microbiol ureases: significance, regulation, and molecular characterization. Microbiol. Rev. 1989; 53(1):85-108.
23. Maeda M, Hindaka M, Nakamura A, et al. Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli. J. Bacteriol. 1994; 176(2): 3432-442
24. Mobley HL, Garner RM, Bauerfeld P. Helicobacter pylori nickel-transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions. Mol.Microbiol. 1995; 16(1): 97-109
25. Collins CM, Dorazio SEF. Bacterial ureases: structure, regulation of expression and role in pathogenesis. Mol. Microbiol. 1993; 9(5):907-913
26. Chen YY, Weaver CA, Mendelsohn DR, et al. Transcriptional regulation of the Streptococcus salivarius 57.I urease operon. J Bacteriol. 1998; 180(21): 5769-5775.
27. Weeks DL, Eskandari S, Scott DR, et al. A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science. 2000; 287(5452): 482-485.
28. Mobley HL, Island MD, Hausinger RP. Molecular biology of ureases. Microbiol. Rev. 1995; 59(3):451-480
29. Jabri E, Carr MB, Hausinger RP, et al. Science. 1995; 268(5213): 998-1004
30. Lee MH, Mulrooney SB, Rener MJ, et al. Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J. Bacteriol. 1992; 174(13): 4324-4330
31. Mulrooney SB, Hausinger RP. Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation. J. Bacteriol. 1990; 172(10): 5837-5843
32. Dorazio SEF, Collins CM. The plasmid-encoded urease gene cluster of the family Enterobacteriaceae is positively regulated by UreR, a member of the AraC family of transcriptional activators. J. Bacteriol. 1993; 175(11): 3459-3467
33. Nicholson EB, Concaugh EA, Foxall PA, et al. Proteus mirabilis urease: transcriptional regulation by UreR. J. Bacteriol. 1993; 175(2): 465-473
34. Sissons CH, Hancock EM. Cutress TW. The source of variation in ureolysis in artificial plaques cultured from human salivary bacteria. Archs Oral Biol. 1988; 33(10): 721-726
35. Sissons CH, Loong PC, Hancock EM. Electrophoretic analysis of ureases in Streptococcus salivarius and in saliva. Oral Microbiol Immunol. 1989; 4(4): 211-218
36. Kopstein J, Wrong OM. The origin and fate of salivary urea and ammonia in man. 1977, Clin. Sci. Molec. Med.52(1): 9-17
37. Sissons CH, Hancock EM. Urease activity in Streptococcus salivarius at low pH. Archs Oral Biol. 1993; 38(6): 507-516
38. Hillman JD, Chen A, Snoep JL. Genetic and physiological analysis of the lethal effect of L-(+)-lactate dehydrogenase deficiency in Streptococcus mutans: complementation by alcohol dehydrogenase from Zymomonas mobilis. Infect. Immun. 1996; 64(10):4319-4323
39. Kubo S, Kubota H, Ohnishi Y, et al. Expression and secretion of an Arthrobacter dextranase in the oral bacterium Streptococcus gordonii. Infect. Immun. 1993; 61(10): 4375-4381
40. Clancy KA, Burne RA. Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity. FEMS Microbiol. Lett. 1997; 151(2):205-211
41. Morou-Bermudez E, Burne RA. Analysis of Urease Expression in Actinomyces naeslundii WVU45. Infect Immun, 2000; 68(12): 6670-6676
42. Chen YY, Clancy KA, Burne RA. Streptococcus salivarius Urease: Genetic and Biochemical Characterization and Expression in a Dental Plaque Streptococcus. Infect Immun, 1996; 64(2): 585-592
43. Bowden GH, Hardie JM, Fillery ED. Antigens from Actinomyces species and their value in identification. J. Dent. Res, 1976; 55: A192-A204
44. Kleiner D. Energy expenditure for cyclic retention of NH3/NH4+ during N2 fixation by Klebsiella pneumoniae. FEBS Lett. 1985; 187(2): 237-239
45. Chen YY, Weaver CA, Burne RA. Dual functions of Streptococcus salivarius urease. J Bacteriol, 2000; 182(16): 4667-4669
46. Morou-Bermudez E, Burne RA. Genetic and physiologic characterization of urease of Actinomyces naeslundii. Infect. Immun, 1999,67(2): 504-512
47. Bradshaw DJ, Mckee AS, Marsh PD. Effects of carbohydrate pulses and pH on population shifts within oral microbial communities in vitro. J Dent Res. 1989, 68(9): 1298-13027.
48. Li YH, Chen YM, Burne RA. Regulation of urease gene expression by Streptococcus salivarius growing in biofilms. Environ Microbiol 2000, 2(2): 169-177
49. Clancy KA, Burne RA. Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity. FEMS Microbiol. Lett, 1997, 151(2): 205-211
50. Chen YY, Burne RA. Analysis of Streptococcus salivarius urease expression using continuous chemostat culture. FEMS Microbiol. Lett, 1996,135(2-3): 223-229
51. Burne RA, Chen YM. The use of continuous flow bioreactors to explore gene expression and physiology of suspended and adherent populations of oral streptococci. Methods Cell Sci. 1998; 20(2):181-190
52. Bowden GH, Hamilton IR.Survival of oral bacteria. Crit Rev Oral Biol Med. 1998; 9(1): 54-85.
53. Sissons CH, Hancock EM, Perinpanayagam HE, et al. The bacteria responsible for ureolysis in artificial dental plaque. Arch Oral Biol. 1988; 33(10): 727-733.
54. Weeks DL, Eskandari S, Scott DR, et al. A H+-gated urea channel: the link between Helicobacter pylori urease and gastric colonization. Science. 2000; 287(2): 482-485.
55. Shu M, Browngardt CM, Chen YY, et al. Role of urease enzymes in stability of a 10-species oral biofilm consortium cultivated in a constant-depth film fermenter. Infect Immun. 2003;71(12): 7188-7192
56. Clancy KA, Burne RA. Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity. FEMS Microbiol Lett. 1997. 151(20): 205-211
57. Clancy KA, Pearson S, Bowen WH, et al. Characterization of recombinant, ureolytic Streptococcus mutans demonstrates an inverse relationship between dental plaque ureolytic capacity and cariogenicity. Infect Immun. 2000;68(5):2621-2629.