Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Jul;63(7):2459–2466. doi: 10.1128/iai.63.7.2459-2466.1995

Characterization of a Tn4351-generated hemin uptake mutant of Porphyromonas gingivalis: evidence for the coordinate regulation of virulence factors by hemin.

C A Genco 1, W Simpson 1, R Y Forng 1, M Egal 1, B M Odusanya 1
PMCID: PMC173328  PMID: 7790057

Abstract

The ability of Porphyromonas gingivalis to acquire iron in the iron-limited environment of the host is crucial to the colonization of this organism. We report here on the isolation and characterization of a transpositional insertion mutant of P. gingivalis A7436 (designated MSM-3) which is defective in the utilization and transport of hemin. P. gingivalis MSM-3 was selected on the basis of its nonpigmented phenotype on anaerobic blood agar following mutagenesis with the Bacteroides fragilis transposon Tn4351. P. gingivalis MSM-3 grew poorly when supplied with hemin as a sole source of iron; however, growth was observed with hemoglobin or inorganic iron. P. gingivalis MSM-3 grown in either hemin-replete or hemin-depleted conditions bound and transported less [14C]hemin or [59Fe]hemin than did the parent strain. At 4 h, P. gingivalis MSM-3 grown in hemin-replete conditions transported only 10,000 pmol of hemin per mg of protein, or 14% of the amount transported by P. gingivalis A7436. Unlike P. gingivalis A7436, hemin binding and transport by P. gingivalis MSM-3 were not tightly regulated by hemin or iron. Examination of P. gingivalis MSM-3 cultures by electron microscopy revealed an overproduction of membrane vesicles, and determination of the dry weight of purified vesicles indicated that P. gingivalis MSM-3 produced twice as much membrane vesicles as did strain A7436. Extracellular vesicles isolated from P. gingivalis MSM-3 also were found to express increased hemolytic and trypsin-like protease activities compared with the parent strain. When inoculated into subcutaneous chambers implanted in mice, P. gingivalis MSM-3 was highly infectious and more invasive than the parent strain, as indicated by secondary lesion formation and death. Taken together, these results indicate that the decreased transport of hemin by P. gingivalis MSM-3 results in the increased expression of several virulence factors which may be coordinately regulated by hemin.

Full Text

The Full Text of this article is available as a PDF (812.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bagg A., Neilands J. B. Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry. 1987 Aug 25;26(17):5471–5477. doi: 10.1021/bi00391a039. [DOI] [PubMed] [Google Scholar]
  2. Barua P. K., Dyer D. W., Neiders M. E. Effect of iron limitation on Bacteroides gingivalis. Oral Microbiol Immunol. 1990 Oct;5(5):263–268. doi: 10.1111/j.1399-302x.1990.tb00423.x. [DOI] [PubMed] [Google Scholar]
  3. Berish S. A., Subbarao S., Chen C. Y., Trees D. L., Morse S. A. Identification and cloning of a fur homolog from Neisseria gonorrhoeae. Infect Immun. 1993 Nov;61(11):4599–4606. doi: 10.1128/iai.61.11.4599-4606.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bourgeau G., Mayrand D. Aggregation of Actinomyces strains by extracellular vesicles produced by Bacteroides gingivalis. Can J Microbiol. 1990 May;36(5):362–365. doi: 10.1139/m90-063. [DOI] [PubMed] [Google Scholar]
  5. Bramanti T. E., Holt S. C., Ebersole J. L., Van Dyke T. Regulation of Porphyromonas gingivalis virulence: hemin limitation effects on the outer membrane protein (OMP) expression and biological activity. J Periodontal Res. 1993 Nov;28(6 Pt 2):464–466. doi: 10.1111/j.1600-0765.1993.tb02103.x. [DOI] [PubMed] [Google Scholar]
  6. Bramanti T. E., Holt S. C. Effect of porphyrins and host iron transport proteins on outer membrane protein expression in Porphyromonas (Bacteroides) gingivalis: identification of a novel 26 kDa hemin-repressible surface protein. Microb Pathog. 1992 Jul;13(1):61–73. doi: 10.1016/0882-4010(92)90032-j. [DOI] [PubMed] [Google Scholar]
  7. Bramanti T. E., Holt S. C. Hemin uptake in Porphyromonas gingivalis: Omp26 is a hemin-binding surface protein. J Bacteriol. 1993 Nov;175(22):7413–7420. doi: 10.1128/jb.175.22.7413-7420.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Braun V., Günther H., Neuss B., Tautz C. Hemolytic activity of Serratia marcescens. Arch Microbiol. 1985 May;141(4):371–376. doi: 10.1007/BF00428852. [DOI] [PubMed] [Google Scholar]
  9. Carlsson J., Höfling J. F., Sundqvist G. K. Degradation of albumin, haemopexin, haptoglobin and transferrin, by black-pigmented Bacteroides species. J Med Microbiol. 1984 Aug;18(1):39–46. doi: 10.1099/00222615-18-1-39. [DOI] [PubMed] [Google Scholar]
  10. Carman R. J., Ramakrishnan M. D., Harper F. H. Hemin levels in culture medium of Porphyromonas (Bacteroides) gingivalis regulate both hemin binding and trypsinlike protease production. Infect Immun. 1990 Dec;58(12):4016–4019. doi: 10.1128/iai.58.12.4016-4019.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cavalieri S. J., Bohach G. A., Snyder I. S. Escherichia coli alpha-hemolysin: characteristics and probable role in pathogenicity. Microbiol Rev. 1984 Dec;48(4):326–343. doi: 10.1128/mr.48.4.326-343.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chu L., Bramanti T. E., Ebersole J. L., Holt S. C. Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization. Infect Immun. 1991 Jun;59(6):1932–1940. doi: 10.1128/iai.59.6.1932-1940.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Collings S., Love D. N. Further studies on some physical and biochemical characteristics of asaccharolytic pigmented Bacteroides of feline origin. J Appl Bacteriol. 1992 Jun;72(6):529–535. doi: 10.1111/j.1365-2672.1992.tb01871.x. [DOI] [PubMed] [Google Scholar]
  14. Cutler C. W., Kalmar J. R., Genco C. A. Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis. Trends Microbiol. 1995 Feb;3(2):45–51. doi: 10.1016/s0966-842x(00)88874-5. [DOI] [PubMed] [Google Scholar]
  15. Genco C. A., Cutler C. W., Kapczynski D., Maloney K., Arnold R. R. A novel mouse model to study the virulence of and host response to Porphyromonas (Bacteroides) gingivalis. Infect Immun. 1991 Apr;59(4):1255–1263. doi: 10.1128/iai.59.4.1255-1263.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Genco C. A., Odusanya B. M., Brown G. Binding and accumulation of hemin in Porphyromonas gingivalis are induced by hemin. Infect Immun. 1994 Jul;62(7):2885–2892. doi: 10.1128/iai.62.7.2885-2892.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Genco C. A. Regulation of hemin and iron transport in Porphyromonas gingivalis. Adv Dent Res. 1995 Feb;9(1):41–47. doi: 10.1177/08959374950090010801. [DOI] [PubMed] [Google Scholar]
  18. Genco C. A., Schifferle R. E., Njoroge T., Forng R. Y., Cutler C. W. Resistance of a Tn4351-generated polysaccharide mutant of Porphyromonas gingivalis to polymorphonuclear leukocyte killing. Infect Immun. 1995 Feb;63(2):393–401. doi: 10.1128/iai.63.2.393-401.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Grenier D. Hemin-binding property of Porphyromonas gingivalis outer membranes. FEMS Microbiol Lett. 1991 Jan 1;61(1):45–49. doi: 10.1016/0378-1097(91)90011-x. [DOI] [PubMed] [Google Scholar]
  20. Hickey E. K., Cianciotto N. P. Cloning and sequencing of the Legionella pneumophila fur gene. Gene. 1994 May 27;143(1):117–121. doi: 10.1016/0378-1119(94)90615-7. [DOI] [PubMed] [Google Scholar]
  21. Hwa V., Shoemaker N. B., Salyers A. A. Direct repeats flanking the Bacteroides transposon Tn4351 are insertion sequence elements. J Bacteriol. 1988 Jan;170(1):449–451. doi: 10.1128/jb.170.1.449-451.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kay H. M., Birss A. J., Smalley J. W. Haemagglutinating and haemolytic activity of the extracellular vesicles of Bacteroides gingivalis W50. Oral Microbiol Immunol. 1990 Oct;5(5):269–274. doi: 10.1111/j.1399-302x.1990.tb00424.x. [DOI] [PubMed] [Google Scholar]
  23. Litwin C. M., Calderwood S. B. Analysis of the complexity of gene regulation by fur in Vibrio cholerae. J Bacteriol. 1994 Jan;176(1):240–248. doi: 10.1128/jb.176.1.240-248.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Litwin C. M., Calderwood S. B. Role of iron in regulation of virulence genes. Clin Microbiol Rev. 1993 Apr;6(2):137–149. doi: 10.1128/cmr.6.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marsh P. D., McDermid A. S., McKee A. S., Baskerville A. The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50. Microbiology. 1994 Apr;140(Pt 4):861–865. doi: 10.1099/00221287-140-4-861. [DOI] [PubMed] [Google Scholar]
  26. Mayrand D., Grenier D. Biological activities of outer membrane vesicles. Can J Microbiol. 1989 Jun;35(6):607–613. doi: 10.1139/m89-097. [DOI] [PubMed] [Google Scholar]
  27. Mayrand D., Holt S. C. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiol Rev. 1988 Mar;52(1):134–152. doi: 10.1128/mr.52.1.134-152.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McKee A. S., McDermid A. S., Baskerville A., Dowsett A. B., Ellwood D. C., Marsh P. D. Effect of hemin on the physiology and virulence of Bacteroides gingivalis W50. Infect Immun. 1986 May;52(2):349–355. doi: 10.1128/iai.52.2.349-355.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mekalanos J. J. Environmental signals controlling expression of virulence determinants in bacteria. J Bacteriol. 1992 Jan;174(1):1–7. doi: 10.1128/jb.174.1.1-7.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Minhas T., Greenman J., Schaffer A. G. The influence of haemin levels on growth and enzyme production by Porphyromonas gingivalis in continuous culture. Microbios. 1993;76(307):105–114. [PubMed] [Google Scholar]
  31. Mukherjee S. The role of crevicular fluid iron in periodontal disease. J Periodontol. 1985 Nov;56(11 Suppl):22–27. doi: 10.1902/jop.1985.56.11s.22. [DOI] [PubMed] [Google Scholar]
  32. Nishina Y., Miyoshi S., Nagase A., Shinoda S. Significant role of an exocellular protease in utilization of heme by Vibrio vulnificus. Infect Immun. 1992 May;60(5):2128–2132. doi: 10.1128/iai.60.5.2128-2132.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Papaioannou S., Marsh P. D., Ivanyi L. The immunogenicity of outer membrane proteins of haemin-depleted Porphyromonas (Bacteroides) gingivalis W50 in periodontal disease. Oral Microbiol Immunol. 1991 Dec;6(6):327–331. doi: 10.1111/j.1399-302x.1991.tb00503.x. [DOI] [PubMed] [Google Scholar]
  34. Progulske-Fox A., Tumwasorn S., Holt S. C. The expression and function of a Bacteroides gingivalis hemagglutinin gene in Escherichia coli. Oral Microbiol Immunol. 1989 Sep;4(3):121–131. doi: 10.1111/j.1399-302x.1989.tb00238.x. [DOI] [PubMed] [Google Scholar]
  35. Shah H. N., Bonnett R., Mateen B., Williams R. A. The porphyrin pigmentation of subspecies of Bacteroides melaninogenicus. Biochem J. 1979 Apr 15;180(1):45–50. doi: 10.1042/bj1800045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shoemaker N. B., Getty C., Gardner J. F., Salyers A. A. Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome. J Bacteriol. 1986 Mar;165(3):929–936. doi: 10.1128/jb.165.3.929-936.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smalley J. W., Birss A. J., McKee A. S., Marsh P. D. Haemin-binding proteins of Porphyromonas gingivalis W50 grown in a chemostat under haemin-limitation. J Gen Microbiol. 1993 Sep;139(9):2145–2150. doi: 10.1099/00221287-139-9-2145. [DOI] [PubMed] [Google Scholar]
  38. Smalley J. W., Birss A. J., McKee A. S., Marsh P. D. Haemin-restriction influences haemin-binding, haemagglutination and protease activity of cells and extracellular membrane vesicles of Porphyromonas gingivalis W50. FEMS Microbiol Lett. 1991 Dec 15;69(1):63–67. doi: 10.1016/0378-1097(91)90647-s. [DOI] [PubMed] [Google Scholar]
  39. Stoebner J. A., Payne S. M. Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae. Infect Immun. 1988 Nov;56(11):2891–2895. doi: 10.1128/iai.56.11.2891-2895.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tai S. S., Lee C. J., Winter R. E. Hemin utilization is related to virulence of Streptococcus pneumoniae. Infect Immun. 1993 Dec;61(12):5401–5405. doi: 10.1128/iai.61.12.5401-5405.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Thomas C. E., Sparling P. F. Identification and cloning of a fur homologue from Neisseria meningitidis. Mol Microbiol. 1994 Feb;11(4):725–737. doi: 10.1111/j.1365-2958.1994.tb00350.x. [DOI] [PubMed] [Google Scholar]
  42. U S., Harper F., Curtis M. A. Haemin inhibits the trypsin-like enzyme activity of Porphyromonas gingivalis W83. FEMS Microbiol Lett. 1990 Oct;60(1-2):169–172. doi: 10.1111/j.1574-6968.1990.tb03883.x. [DOI] [PubMed] [Google Scholar]
  43. Wooldridge K. G., Williams P. H. Iron uptake mechanisms of pathogenic bacteria. FEMS Microbiol Rev. 1993 Nov;12(4):325–348. doi: 10.1111/j.1574-6976.1993.tb00026.x. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES