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. 1990 Jul;58(7):2320–2328. doi: 10.1128/iai.58.7.2320-2328.1990

Penetration and intracellular growth of Brucella abortus in nonphagocytic cells in vitro.

P G Detilleux 1, B L Deyoe 1, N F Cheville 1
PMCID: PMC258815  PMID: 2114362

Abstract

In pregnant ruminants, Brucella abortus localizes and replicates within the rough endoplasmic reticulum of trophoblastic epithelial cells. In this study, Vero cells were exposed to B. abortus to investigate its internalization and intracellular growth in nonphagocytic cells. A new double-fluorescence staining procedure to discriminate between extracellular and intracellular bacteria was developed. Studies with the double-fluorescence staining procedure and quantitative bacteriologic culture of disrupted host cells showed that various B. abortus strains replicated within Vero cells, including smooth virulent (strains 2308S and 544), smooth attenuated (strain 19), and rough (strains 45/20 and 2308R) strains. Rough brucellae were more adherent and entered a greater number of Vero cells. Intracellular replication occurred in a larger percentage of cells with smooth virulent (2308S and 544) strains than with smooth attenuated (19) or rough (45/20 and 2308R) strains. Differences in adhesiveness and invasiveness were correlated to hydrophobicity of the organism, as measured by hydrocarbon adherence. Ultrastructurally, intracellular smooth (2308S) and rough (45/20) brucellae were consistently found within cisternae of the rough endoplasmic reticulum and nuclear envelope. The results suggest that transfer to the rough endoplasmic reticulum is the limiting step in the infection of nonphagocytic cells by B. abortus.

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Selected References

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  1. Anderson T. D., Cheville N. F., Meador V. P. Pathogenesis of placentitis in the goat inoculated with Brucella abortus. II. Ultrastructural studies. Vet Pathol. 1986 May;23(3):227–239. doi: 10.1177/030098588602300302. [DOI] [PubMed] [Google Scholar]
  2. Anderson T. D., Cheville N. F. Ultrastructural morphometric analysis of Brucella abortus-infected trophoblasts in experimental placentitis. Bacterial replication occurs in rough endoplasmic reticulum. Am J Pathol. 1986 Aug;124(2):226–237. [PMC free article] [PubMed] [Google Scholar]
  3. Barrow P. A., Lovell M. A. Invasion of Vero cells by Salmonella species. J Med Microbiol. 1989 Jan;28(1):59–67. doi: 10.1099/00222615-28-1-59. [DOI] [PubMed] [Google Scholar]
  4. Bukholm G., Johansen B. V., Namork E., Lassen J. Bacterial adhesiveness and invasiveness in cell culture monolayer. Acta Pathol Microbiol Immunol Scand B. 1982 Dec;90(6):403–408. doi: 10.1111/j.1699-0463.1982.tb00138.x. [DOI] [PubMed] [Google Scholar]
  5. Corbeil L. B., Blau K., Inzana T. J., Nielsen K. H., Jacobson R. H., Corbeil R. R., Winter A. J. Killing of Brucella abortus by bovine serum. Infect Immun. 1988 Dec;56(12):3251–3261. doi: 10.1128/iai.56.12.3251-3261.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Corbel M. J. The effect of synthetic polynucleotides on the intracellular growth of Brucella abortus in vitro and in vivo. Br Vet J. 1980 Mar-Apr;136(2):175–181. doi: 10.1016/s0007-1935(17)32341-2. [DOI] [PubMed] [Google Scholar]
  7. Detilleux P. G., Cheville N. F., Deyoe B. L. Pathogenesis of Brucella abortus in chicken embryos. Vet Pathol. 1988 Mar;25(2):138–146. doi: 10.1177/030098588802500206. [DOI] [PubMed] [Google Scholar]
  8. Giannella R. A., Washington O., Gemski P., Formal S. B. Invasion of HeLa cells by Salmonella typhimurium: a model for study of invasiveness of Salmonella. J Infect Dis. 1973 Jul;128(1):69–75. doi: 10.1093/infdis/128.1.69. [DOI] [PubMed] [Google Scholar]
  9. Glavin F. L., Winn W. C., Jr, Craighead J. E. Ultrastructure of lung in Legionnaires' disease. Observations of three biopsies done during the Vermont epidemic. Ann Intern Med. 1979 Apr;90(4):555–559. doi: 10.7326/0003-4819-90-4-555. [DOI] [PubMed] [Google Scholar]
  10. HOLLAND J. J., PICKETT M. J. Intracellular behavior of Brucella variants in chick embryo cells in tissue culture. Proc Soc Exp Biol Med. 1956 Dec;93(3):476–479. doi: 10.3181/00379727-93-22792. [DOI] [PubMed] [Google Scholar]
  11. Hale T. L., Bonventre P. F. Shigella infection of Henle intestinal epithelial cells: role of the bacterium. Infect Immun. 1979 Jun;24(3):879–886. doi: 10.1128/iai.24.3.879-886.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harmon B. G., Adams L. G., Frey M. Survival of rough and smooth strains of Brucella abortus in bovine mammary gland macrophages. Am J Vet Res. 1988 Jul;49(7):1092–1097. [PubMed] [Google Scholar]
  13. Hatten B. A., Sulkin S. E. Intracellular Production of Brucella L Forms I. Recovery of L Forms from Tissue Culture Cells Infected with Brucella abortus. J Bacteriol. 1966 Jan;91(1):285–296. doi: 10.1128/jb.91.1.285-296.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Heesemann J., Laufs R. Double immunofluorescence microscopic technique for accurate differentiation of extracellularly and intracellularly located bacteria in cell culture. J Clin Microbiol. 1985 Aug;22(2):168–175. doi: 10.1128/jcm.22.2.168-175.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Horwitz M. A. Formation of a novel phagosome by the Legionnaires' disease bacterium (Legionella pneumophila) in human monocytes. J Exp Med. 1983 Oct 1;158(4):1319–1331. doi: 10.1084/jem.158.4.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jones G. W., Richardson L. A., Uhlman D. The invasion of HeLa cells by Salmonella typhimurium: reversible and irreversible bacterial attachment and the role of bacterial motility. J Gen Microbiol. 1981 Dec;127(2):351–360. doi: 10.1099/00221287-127-2-351. [DOI] [PubMed] [Google Scholar]
  17. Kihlström E., Edebo L. Association of viable and inactivated Salmonella typhimurium 395 MS and MR 10 with HeLa cells. Infect Immun. 1976 Oct;14(4):851–857. doi: 10.1128/iai.14.4.851-857.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kihlström E. Infection of HeLa cells with Salmonella typhimurium 395 MS and MR10 bacteria. Infect Immun. 1977 Aug;17(2):290–295. doi: 10.1128/iai.17.2.290-295.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kihlström E., Latkovic S. Ultrastructural studies on the interaction between Salmonella typhimurium 395 M and HeLa cells. Infect Immun. 1978 Dec;22(3):804–809. doi: 10.1128/iai.22.3.804-809.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kreutzer D. L., Robertson D. C. Surface macromolecules and virulence in intracellular parasitism: comparison of cell envelope components of smooth and rough strains of Brucella abortus. Infect Immun. 1979 Mar;23(3):819–828. doi: 10.1128/iai.23.3.819-828.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  22. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Meador V. P., Deyoe B. L. Intracellular localization of Brucella abortus in bovine placenta. Vet Pathol. 1989 Nov;26(6):513–515. doi: 10.1177/030098588902600609. [DOI] [PubMed] [Google Scholar]
  24. Mehlman I. J., Eide E. L., Sanders A. C., Fishbein M., Aulisio C. C. Methodology for recognition of invasive potential of Escherichia coli. J Assoc Off Anal Chem. 1977 May;60(3):546–562. [PubMed] [Google Scholar]
  25. Oldham L. J., Rodgers F. G. Adhesion, penetration and intracellular replication of Legionella pneumophila: an in vitro model of pathogenesis. J Gen Microbiol. 1985 Apr;131(4):697–706. doi: 10.1099/00221287-131-4-697. [DOI] [PubMed] [Google Scholar]
  26. RICHARDSON M., HOLT J. N. MULTIPLICATION OF BRUCELLA IN CULTURED LYMPHOID AND NONLYMPHOID CELLS. J Bacteriol. 1964 Oct;88:1163–1168. doi: 10.1128/jb.88.4.1163-1168.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Richardson M. PARASITIZATION IN VITRO OF BOVINE CELLS BY BRUCELLA ABORTUS. J Bacteriol. 1959 Dec;78(6):769–777. doi: 10.1128/jb.78.6.769-777.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Riley L. K., Robertson D. C. Brucellacidal activity of human and bovine polymorphonuclear leukocyte granule extracts against smooth and rough strains of Brucella abortus. Infect Immun. 1984 Oct;46(1):231–236. doi: 10.1128/iai.46.1.231-236.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Riley L. K., Robertson D. C. Ingestion and intracellular survival of Brucella abortus in human and bovine polymorphonuclear leukocytes. Infect Immun. 1984 Oct;46(1):224–230. doi: 10.1128/iai.46.1.224-230.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Roux J., Arnaud-Bosq C. La multiplication intracellulaire de Brucella (Théorie pour un modèle expérimental) Ann Microbiol (Paris) 1975 May-Jun;126A(4):461–481. [PubMed] [Google Scholar]
  31. SHEPARD C. C. Nonacid-fast bacteria and HeLa cells: their uptake and subsequent intracellular growth. J Bacteriol. 1959 Jun;77(6):701–714. doi: 10.1128/jb.77.6.701-714.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sansonetti P. J., Ryter A., Clerc P., Maurelli A. T., Mounier J. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun. 1986 Feb;51(2):461–469. doi: 10.1128/iai.51.2.461-469.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schiemann D. A., Crane M. R., Swanz P. J. Surface properties of Yersinia species and epithelial cell interactions in vitro by a method measuring total associated, attached and intracellular bacteria. J Med Microbiol. 1987 Nov;24(3):205–218. doi: 10.1099/00222615-24-3-205. [DOI] [PubMed] [Google Scholar]
  34. Sperry J. F., Robertson D. C. Inhibition of growth by erythritol catabolism in Brucella abortus. J Bacteriol. 1975 Oct;124(1):391–397. doi: 10.1128/jb.124.1.391-397.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vesikari T., Bromirska J., Mäki M. Enhancement of invasiveness of Yersinia enterocolitica and Escherichia coli in HEp-2 cells by centrifugation. Infect Immun. 1982 May;36(2):834–836. doi: 10.1128/iai.36.2.834-836.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. WHITE P. G., WILSON J. B. Differentiation of smooth and nonsmooth colonies of Brucellae. J Bacteriol. 1951 Feb;61(2):239–240. doi: 10.1128/jb.61.2.239-240.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]

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