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. 1992 Nov;60(11):4578–4585. doi: 10.1128/iai.60.11.4578-4585.1992

Uptake and intracellular survival of Bordetella pertussis in human macrophages.

R L Friedman 1, K Nordensson 1, L Wilson 1, E T Akporiaye 1, D E Yocum 1
PMCID: PMC258205  PMID: 1398970

Abstract

Recent reports have demonstrated that Bordetella pertussis has invasive behavior in vivo and in vitro. In this study, we investigated the ability of a virulent strain, avirulent mutants, and mutants deficient in specific virulence factors to enter and survive intracellularly in human macrophages in vitro. Uptake of virulent B. pertussis was dose dependent and occurred in the absence of serum or specific antibody, with entry occurring via a microfilament-dependent phagocytic process. The virulent wild-type parental strain was internalized and persisted intracellularly over the 3 days of experiments, as determined by transmission electron microscopy and by recovery of viable plate counts. This is the first report of long-term survival of B. pertussis in human macrophages. Avirulent mutants entered macrophages, but at only an average of 1.5% of virulent parental levels, and did not survive intracellularly. Mutants which did not express adenylate cyclase toxin, filamentous hemagglutinin, or pertussis toxin had decreased abilities to enter and to survive inside macrophages. The results suggest that the internalization process, as well as intracellular survival, is virulence dependent and that mutations which inactivate expression of virulence factors may affect both. The ability of B. pertussis to enter and persist inside macrophages may be important not only for survival of the bacteria but also in the pathogenesis of whooping cough.

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  1. Adamson P. C., Wu T. C., Meade B. D., Rubin M., Manclark C. R., Pizzo P. A. Pertussis in a previously immunized child with human immunodeficiency virus infection. J Pediatr. 1989 Oct;115(4):589–592. doi: 10.1016/s0022-3476(89)80288-4. [DOI] [PubMed] [Google Scholar]
  2. Becker E. L., Kermode J. C., Naccache P. H., Yassin R., Munoz J. J., Marsh M. L., Huang C. K., Sha'afi R. I. Pertussis toxin as a probe of neutrophil activation. Fed Proc. 1986 Jun;45(7):2151–2155. [PubMed] [Google Scholar]
  3. Bromberg K., Tannis G., Steiner P. Detection of Bordetella pertussis associated with the alveolar macrophages of children with human immunodeficiency virus infection. Infect Immun. 1991 Dec;59(12):4715–4719. doi: 10.1128/iai.59.12.4715-4719.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bullock W. E., Wright S. D. Role of the adherence-promoting receptors, CR3, LFA-1, and p150,95, in binding of Histoplasma capsulatum by human macrophages. J Exp Med. 1987 Jan 1;165(1):195–210. doi: 10.1084/jem.165.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  6. CRAWFORD J. G., FISHEL C. W. Growth of Bordetella pertussis in tissue culture. J Bacteriol. 1959 Apr;77(4):465–474. doi: 10.1128/jb.77.4.465-474.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Charles I. G., Dougan G., Pickard D., Chatfield S., Smith M., Novotny P., Morrissey P., Fairweather N. F. Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis. Proc Natl Acad Sci U S A. 1989 May;86(10):3554–3558. doi: 10.1073/pnas.86.10.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheers C., Gray D. F. Macrophage behaviour during the complaisant phase of murine pertussis. Immunology. 1969 Dec;17(6):875–887. [PMC free article] [PubMed] [Google Scholar]
  9. Confer D. L., Eaton J. W. Phagocyte impotence caused by an invasive bacterial adenylate cyclase. Science. 1982 Sep 3;217(4563):948–950. doi: 10.1126/science.6287574. [DOI] [PubMed] [Google Scholar]
  10. Cookson B. T., Berg D. E., Goldman W. E. Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes. J Bacteriol. 1990 Apr;172(4):1681–1687. doi: 10.1128/jb.172.4.1681-1687.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Coote J. G. Antigenic switching and pathogenicity: environmental effects on virulence gene expression in Bordetella pertussis. J Gen Microbiol. 1991 Nov;137(11):2493–2503. doi: 10.1099/00221287-137-11-2493. [DOI] [PubMed] [Google Scholar]
  12. Domenighini M., Relman D., Capiau C., Falkow S., Prugnola A., Scarlato V., Rappuoli R. Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor. Mol Microbiol. 1990 May;4(5):787–800. doi: 10.1111/j.1365-2958.1990.tb00649.x. [DOI] [PubMed] [Google Scholar]
  13. Ewanowich C. A., Melton A. R., Weiss A. A., Sherburne R. K., Peppler M. S. Invasion of HeLa 229 cells by virulent Bordetella pertussis. Infect Immun. 1989 Sep;57(9):2698–2704. doi: 10.1128/iai.57.9.2698-2704.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ewanowich C. A., Sherburne R. K., Man S. F., Peppler M. S. Bordetella parapertussis invasion of HeLa 229 cells and human respiratory epithelial cells in primary culture. Infect Immun. 1989 Apr;57(4):1240–1247. doi: 10.1128/iai.57.4.1240-1247.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Finlay B. B., Falkow S. Comparison of the invasion strategies used by Salmonella cholerae-suis, Shigella flexneri and Yersinia enterocolitica to enter cultured animal cells: endosome acidification is not required for bacterial invasion or intracellular replication. Biochimie. 1988 Aug;70(8):1089–1099. doi: 10.1016/0300-9084(88)90271-4. [DOI] [PubMed] [Google Scholar]
  16. Friedman R. L., Fiederlein R. L., Glasser L., Galgiani J. N. Bordetella pertussis adenylate cyclase: effects of affinity-purified adenylate cyclase on human polymorphonuclear leukocyte functions. Infect Immun. 1987 Jan;55(1):135–140. doi: 10.1128/iai.55.1.135-140.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Friedman R. L. Pertussis: the disease and new diagnostic methods. Clin Microbiol Rev. 1988 Oct;1(4):365–376. doi: 10.1128/cmr.1.4.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Galgiani J. N., Hewlett E. L., Friedman R. L. Effects of adenylate cyclase toxin from Bordetella pertussis on human neutrophil interactions with Coccidioides immitis and Staphylococcus aureus. Infect Immun. 1988 Apr;56(4):751–755. doi: 10.1128/iai.56.4.751-755.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goldman W. E., Klapper D. G., Baseman J. B. Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells. Infect Immun. 1982 May;36(2):782–794. doi: 10.1128/iai.36.2.782-794.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gray D. F., Cheers C. The steady state in cellular immunity. II. Immunological complaisance in murine pertussis. Aust J Exp Biol Med Sci. 1967 Aug;45(4):417–426. doi: 10.1038/icb.1967.40. [DOI] [PubMed] [Google Scholar]
  21. Gross R., Aricò B., Rappuoli R. Genetics of pertussis toxin. Mol Microbiol. 1989 Jan;3(1):119–124. doi: 10.1111/j.1365-2958.1989.tb00111.x. [DOI] [PubMed] [Google Scholar]
  22. Hand W. L., King-Thompson N. L. Contrasts between phagocyte antibiotic uptake and subsequent intracellular bactericidal activity. Antimicrob Agents Chemother. 1986 Jan;29(1):135–140. doi: 10.1128/aac.29.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Isberg R. R., Leong J. M. Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell. 1990 Mar 9;60(5):861–871. doi: 10.1016/0092-8674(90)90099-z. [DOI] [PubMed] [Google Scholar]
  24. Laoide B. M., Ullmann A. Virulence dependent and independent regulation of the Bordetella pertussis cya operon. EMBO J. 1990 Apr;9(4):999–1005. doi: 10.1002/j.1460-2075.1990.tb08202.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lee C. K., Roberts A. L., Finn T. M., Knapp S., Mekalanos J. J. A new assay for invasion of HeLa 229 cells by Bordetella pertussis: effects of inhibitors, phenotypic modulation, and genetic alterations. Infect Immun. 1990 Aug;58(8):2516–2522. doi: 10.1128/iai.58.8.2516-2522.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Leininger E., Ewanowich C. A., Bhargava A., Peppler M. S., Kenimer J. G., Brennan M. J. Comparative roles of the Arg-Gly-Asp sequence present in the Bordetella pertussis adhesins pertactin and filamentous hemagglutinin. Infect Immun. 1992 Jun;60(6):2380–2385. doi: 10.1128/iai.60.6.2380-2385.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Leininger E., Roberts M., Kenimer J. G., Charles I. G., Fairweather N., Novotny P., Brennan M. J. Pertactin, an Arg-Gly-Asp-containing Bordetella pertussis surface protein that promotes adherence of mammalian cells. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):345–349. doi: 10.1073/pnas.88.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Leusch M. S., Paulaitis S., Friedman R. L. Adenylate cyclase toxin of Bordetella pertussis: production, purification, and partial characterization. Infect Immun. 1990 Nov;58(11):3621–3626. doi: 10.1128/iai.58.11.3621-3626.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lowrie D. B., Aber V. R., Jackett P. S. Phagosome-lysosome fusion and cyclic adenosine 3':5'-monophosphate in macrophages infected with Mycobacterium microti, Mycobacterium bovis BCG or Mycobacterium lepraemurium. J Gen Microbiol. 1979 Feb;110(2):431–441. doi: 10.1099/00221287-110-2-431. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Miller J. F., Mekalanos J. J., Falkow S. Coordinate regulation and sensory transduction in the control of bacterial virulence. Science. 1989 Feb 17;243(4893):916–922. doi: 10.1126/science.2537530. [DOI] [PubMed] [Google Scholar]
  32. Miller J. F., Roy C. R., Falkow S. Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli. J Bacteriol. 1989 Nov;171(11):6345–6348. doi: 10.1128/jb.171.11.6345-6348.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mouallem M., Farfel Z., Hanski E. Bordetella pertussis adenylate cyclase toxin: intoxication of host cells by bacterial invasion. Infect Immun. 1990 Nov;58(11):3759–3764. doi: 10.1128/iai.58.11.3759-3764.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Moulder J. W. Comparative biology of intracellular parasitism. Microbiol Rev. 1985 Sep;49(3):298–337. doi: 10.1128/mr.49.3.298-337.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ng V. L., York M., Hadley W. K. Unexpected isolation of Bordetella pertussis from patients with acquired immunodeficiency syndrome. J Clin Microbiol. 1989 Feb;27(2):337–338. doi: 10.1128/jcm.27.2.337-338.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Payne N. R., Horwitz M. A. Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J Exp Med. 1987 Nov 1;166(5):1377–1389. doi: 10.1084/jem.166.5.1377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pittman M. The concept of pertussis as a toxin-mediated disease. Pediatr Infect Dis. 1984 Sep-Oct;3(5):467–486. doi: 10.1097/00006454-198409000-00019. [DOI] [PubMed] [Google Scholar]
  38. Relman D. A., Domenighini M., Tuomanen E., Rappuoli R., Falkow S. Filamentous hemagglutinin of Bordetella pertussis: nucleotide sequence and crucial role in adherence. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2637–2641. doi: 10.1073/pnas.86.8.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Relman D., Tuomanen E., Falkow S., Golenbock D. T., Saukkonen K., Wright S. D. Recognition of a bacterial adhesion by an integrin: macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell. 1990 Jun 29;61(7):1375–1382. doi: 10.1016/0092-8674(90)90701-f. [DOI] [PubMed] [Google Scholar]
  40. Roberts M., Fairweather N. F., Leininger E., Pickard D., Hewlett E. L., Robinson A., Hayward C., Dougan G., Charles I. G. Construction and characterization of Bordetella pertussis mutants lacking the vir-regulated P.69 outer membrane protein. Mol Microbiol. 1991 Jun;5(6):1393–1404. doi: 10.1111/j.1365-2958.1991.tb00786.x. [DOI] [PubMed] [Google Scholar]
  41. Roy C. R., Falkow S. Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon. J Bacteriol. 1991 Apr;173(7):2385–2392. doi: 10.1128/jb.173.7.2385-2392.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sato Y., Cowell J. L., Sato H., Burstyn D. G., Manclark C. R. Separation and purification of the hemagglutinins from Bordetella pertussis. Infect Immun. 1983 Jul;41(1):313–320. doi: 10.1128/iai.41.1.313-320.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sato Y., Izumiya K., Sato H., Cowell J. L., Manclark C. R. Role of antibody to leukocytosis-promoting factor hemagglutinin and to filamentous hemagglutinin in immunity to pertussis. Infect Immun. 1981 Mar;31(3):1223–1231. doi: 10.1128/iai.31.3.1223-1231.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Saukkonen K., Cabellos C., Burroughs M., Prasad S., Tuomanen E. Integrin-mediated localization of Bordetella pertussis within macrophages: role in pulmonary colonization. J Exp Med. 1991 May 1;173(5):1143–1149. doi: 10.1084/jem.173.5.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Scarlato V., Prugnola A., Aricó B., Rappuoli R. Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6753–6757. doi: 10.1073/pnas.87.17.6753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schlegel R., Dickson R. B., Willingham M. C., Pastan I. H. Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha 2-macroglobulin. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2291–2295. doi: 10.1073/pnas.79.7.2291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schlesinger L. S., Horwitz M. A. Phagocytosis of leprosy bacilli is mediated by complement receptors CR1 and CR3 on human monocytes and complement component C3 in serum. J Clin Invest. 1990 Apr;85(4):1304–1314. doi: 10.1172/JCI114568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Small P. L., Isberg R. R., Falkow S. Comparison of the ability of enteroinvasive Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Yersinia enterocolitica to enter and replicate within HEp-2 cells. Infect Immun. 1987 Jul;55(7):1674–1679. doi: 10.1128/iai.55.7.1674-1679.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Spangrude G. J., Sacchi F., Hill H. R., Van Epps D. E., Daynes R. A. Inhibition of lymphocyte and neutrophil chemotaxis by pertussis toxin. J Immunol. 1985 Dec;135(6):4135–4143. [PubMed] [Google Scholar]
  50. Steed L. L., Akporiaye E. T., Friedman R. L. Bordetella pertussis induces respiratory burst activity in human polymorphonuclear leukocytes. Infect Immun. 1992 May;60(5):2101–2105. doi: 10.1128/iai.60.5.2101-2105.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Steed L. L., Setareh M., Friedman R. L. Intracellular survival of virulent Bordetella pertussis in human polymorphonuclear leukocytes. J Leukoc Biol. 1991 Oct;50(4):321–330. doi: 10.1002/jlb.50.4.321. [DOI] [PubMed] [Google Scholar]
  52. Steven A. C., Bisher M. E., Trus B. L., Thomas D., Zhang J. M., Cowell J. L. Helical structure of Bordetella pertussis fimbriae. J Bacteriol. 1986 Sep;167(3):968–974. doi: 10.1128/jb.167.3.968-974.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stibitz S., Weiss A. A., Falkow S. Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir. J Bacteriol. 1988 Jul;170(7):2904–2913. doi: 10.1128/jb.170.7.2904-2913.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tuomanen E., Weiss A. Characterization of two adhesins of Bordetella pertussis for human ciliated respiratory-epithelial cells. J Infect Dis. 1985 Jul;152(1):118–125. doi: 10.1093/infdis/152.1.118. [DOI] [PubMed] [Google Scholar]
  55. Urisu A., Cowell J. L., Manclark C. R. Filamentous hemagglutinin has a major role in mediating adherence of Bordetella pertussis to human WiDr cells. Infect Immun. 1986 Jun;52(3):695–701. doi: 10.1128/iai.52.3.695-701.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun. 1983 Oct;42(1):33–41. doi: 10.1128/iai.42.1.33-41.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weiss A. A., Hewlett E. L. Virulence factors of Bordetella pertussis. Annu Rev Microbiol. 1986;40:661–686. doi: 10.1146/annurev.mi.40.100186.003305. [DOI] [PubMed] [Google Scholar]
  58. Weissmann G., Goldstein I., Hoffstein S., Chauvet G., Robineaux R. Yin/Yang modulation of lysosomal enzyme release from polymorphonuclear leukocytes by cyclic nucleotides. Ann N Y Acad Sci. 1975 Jun 13;256:222–232. doi: 10.1111/j.1749-6632.1975.tb36049.x. [DOI] [PubMed] [Google Scholar]
  59. Willems R., Paul A., van der Heide H. G., ter Avest A. R., Mooi F. R. Fimbrial phase variation in Bordetella pertussis: a novel mechanism for transcriptional regulation. EMBO J. 1990 Sep;9(9):2803–2809. doi: 10.1002/j.1460-2075.1990.tb07468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wilson M. E., Pearson R. D. Roles of CR3 and mannose receptors in the attachment and ingestion of Leishmania donovani by human mononuclear phagocytes. Infect Immun. 1988 Feb;56(2):363–369. doi: 10.1128/iai.56.2.363-369.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Woods D. E., Franklin R., Cryz S. J., Jr, Ganss M., Peppler M., Ewanowich C. Development of a rat model for respiratory infection with Bordetella pertussis. Infect Immun. 1989 Apr;57(4):1018–1024. doi: 10.1128/iai.57.4.1018-1024.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhang Y. L., Sekura R. D. Purification and characterization of the heat-labile toxin of Bordetella pertussis. Infect Immun. 1991 Oct;59(10):3754–3759. doi: 10.1128/iai.59.10.3754-3759.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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