Growth of Francisella tularensis LVS in macrophages: the acidic intracellular compartment provides essential iron required for growth (original) (raw)

Abstract

Murine macrophages supported exponential intracellular growth of Francisella tularensis LVS in vitro with a doubling time of 4 to 6 h. LVS was internalized and remained in a vacuolar compartment throughout its growth cycle. The importance of endosome acidification to intracellular growth of this bacterium was assessed by treatment of LVS-infected macrophages with several different lysosomotropic agents (chloroquine, NH4Cl, and ouabain). Regardless of the agent used or its mechanism of action, macrophages treated with agents that blocked endosome acidification no longer supported replication of LVS. Over several experiments for each lysosomotropic agent, the number of CFU of LVS recovered from treated macrophage cultures was equivalent to the input inoculum (approximately 10(4) CFU) at 72 h. In contrast, over 10(8) CFU was consistently recovered from untreated cultures. Pretreatment of macrophages with these endosome acidification inhibitors did not alter their ingestion of bacteria. Further, the effects of the inhibitors were completely reversible: inhibitor-pretreated LVS-infected macrophages washed free of the agent and cultured in medium fully supported LVS growth over 72 h. Endosome acidification is an important cellular event essential for release of iron from transferrin. The growth-inhibitory effects of both chloroquine and NH4Cl were completely reversed by addition of ferric PPi, a transferrin-independent iron source, at a neutral pH but not by addition of excess holotransferrin. Thus, intracellular localization in an acidic vesicle which facilitates the availability of iron essential for Francisella growth is a survival tactic of this bacterium, and iron depletion is one mechanism that macrophages use to inhibit its growth.

Full Text

The Full Text of this article is available as a PDF (598.6 KB).

Selected References

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

  1. Anthony L. D., Burke R. D., Nano F. E. Growth of Francisella spp. in rodent macrophages. Infect Immun. 1991 Sep;59(9):3291–3296. doi: 10.1128/iai.59.9.3291-3296.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong J. A., Hart P. D. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J Exp Med. 1971 Sep 1;134(3 Pt 1):713–740. doi: 10.1084/jem.134.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker C. N., Hollis D. G., Thornsberry C. Antimicrobial susceptibility testing of Francisella tularensis with a modified Mueller-Hinton broth. J Clin Microbiol. 1985 Aug;22(2):212–215. doi: 10.1128/jcm.22.2.212-215.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banerjee-Bhatnagar N., Frasch C. E. Expression of Neisseria meningitidis iron-regulated outer membrane proteins, including a 70-kilodalton transferrin receptor, and their potential for use as vaccines. Infect Immun. 1990 Sep;58(9):2875–2881. doi: 10.1128/iai.58.9.2875-2881.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benjamin W. H., Jr, Turnbough C. L., Jr, Posey B. S., Briles D. E. The ability of Salmonella typhimurium to produce the siderophore enterobactin is not a virulence factor in mouse typhoid. Infect Immun. 1985 Nov;50(2):392–397. doi: 10.1128/iai.50.2.392-397.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown M. R., Williams P. The influence of environment on envelope properties affecting survival of bacteria in infections. Annu Rev Microbiol. 1985;39:527–556. doi: 10.1146/annurev.mi.39.100185.002523. [DOI] [PubMed] [Google Scholar]
  7. Byrd T. F., Horwitz M. A. Chloroquine inhibits the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. A potential new mechanism for the therapeutic effect of chloroquine against intracellular pathogens. J Clin Invest. 1991 Jul;88(1):351–357. doi: 10.1172/JCI115301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cartwright G. E., Lauritsen M. A., Humphreys S., Jones P. J., Merrill I. M., Wintrobe M. M. THE ANEMIA OF INFECTION. II. THE EXPERIMENTAL PRODUCTION OF HYPOFERREMIA AND ANEMIA IN DOGS. J Clin Invest. 1946 Jan;25(1):81–86. doi: 10.1172/JCI101691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang K. P., Dwyer D. M. Multiplication of a human parasite (Leishmania donovani) in phagolysosomes of hamster macrophages in vitro. Science. 1976 Aug 20;193(4254):678–680. doi: 10.1126/science.948742. [DOI] [PubMed] [Google Scholar]
  10. Channon J. Y., Blackwell J. M. A study of the sensitivity of Leishmania donovani promastigotes and amastigotes to hydrogen peroxide. II. Possible mechanisms involved in protective H2O2 scavenging. Parasitology. 1985 Oct;91(Pt 2):207–217. doi: 10.1017/s0031182000057310. [DOI] [PubMed] [Google Scholar]
  11. Ewing E. P., Jr, Takeuchi A., Shirai A., Osterman J. V. Experimental infection of mouse peritoneal mesothelium with scrub typhus rickettsiae: an ultrastructural study. Infect Immun. 1978 Mar;19(3):1068–1075. doi: 10.1128/iai.19.3.1068-1075.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fortier A. H., Polsinelli T., Green S. J., Nacy C. A. Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules. Infect Immun. 1992 Mar;60(3):817–825. doi: 10.1128/iai.60.3.817-825.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fortier A. H., Slayter M. V., Ziemba R., Meltzer M. S., Nacy C. A. Live vaccine strain of Francisella tularensis: infection and immunity in mice. Infect Immun. 1991 Sep;59(9):2922–2928. doi: 10.1128/iai.59.9.2922-2928.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldberg D. E., Slater A. F., Cerami A., Henderson G. B. Hemoglobin degradation in the malaria parasite Plasmodium falciparum: an ordered process in a unique organelle. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2931–2935. doi: 10.1073/pnas.87.8.2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hale T. L. Invasion of epithelial cells by shigellae. Ann Inst Pasteur Microbiol. 1986 May-Jun;137A(3):311–314. doi: 10.1016/s0769-2609(86)80040-0. [DOI] [PubMed] [Google Scholar]
  16. Izhar M., Nuchamowitz Y., Mirelman D. Adherence of Shigella flexneri to guinea pig intestinal cells is mediated by a mucosal adhesion. Infect Immun. 1982 Mar;35(3):1110–1118. doi: 10.1128/iai.35.3.1110-1118.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones T. C., Hirsch J. G. The interaction between Toxoplasma gondii and mammalian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J Exp Med. 1972 Nov 1;136(5):1173–1194. doi: 10.1084/jem.136.5.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lawlor K. M., Daskaleros P. A., Robinson R. E., Payne S. M. Virulence of iron transport mutants of Shigella flexneri and utilization of host iron compounds. Infect Immun. 1987 Mar;55(3):594–599. doi: 10.1128/iai.55.3.594-599.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Long G. W., Oprandy J. J., Narayanan R. B., Fortier A. H., Porter K. R., Nacy C. A. Detection of Francisella tularensis in blood by polymerase chain reaction. J Clin Microbiol. 1993 Jan;31(1):152–154. doi: 10.1128/jcm.31.1.152-154.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Loo V. G., Lalonde R. G. Role of iron in intracellular growth of Trypanosoma cruzi. Infect Immun. 1984 Sep;45(3):726–730. doi: 10.1128/iai.45.3.726-730.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Löfgren S., Tärnvik A., Thore M., Carlsson J. A wild and an attenuated strain of Francisella tularensis differ in susceptibility to hypochlorous acid: a possible explanation of their different handling by polymorphonuclear leukocytes. Infect Immun. 1984 Feb;43(2):730–734. doi: 10.1128/iai.43.2.730-734.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Narayanan R. B., Drabick J. J., Williams J. C., Fortier A. H., Meltzer M. S., Sadoff J. C., Bolt C. R., Nacy C. A. Immunotherapy of tularemia: characterization of a monoclonal antibody reactive with Francisella tularensis. J Leukoc Biol. 1993 Jan;53(1):112–116. doi: 10.1002/jlb.53.1.112. [DOI] [PubMed] [Google Scholar]
  23. Rao K., van Renswoude J., Kempf C., Klausner R. D. Separation of Fe+3 from transferrin in endocytosis. Role of the acidic endosome. FEBS Lett. 1983 Aug 22;160(1-2):213–216. doi: 10.1016/0014-5793(83)80969-7. [DOI] [PubMed] [Google Scholar]
  24. Rhinehart-Jones T. R., Fortier A. H., Elkins K. L. Transfer of immunity against lethal murine Francisella infection by specific antibody depends on host gamma interferon and T cells. Infect Immun. 1994 Aug;62(8):3129–3137. doi: 10.1128/iai.62.8.3129-3137.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. TIGERTT W. D. Soviet viable Pasteurella tularensis vaccines. A review of selected articles. Bacteriol Rev. 1962 Sep;26:354–373. doi: 10.1128/br.26.3.354-373.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tilney L. G., Portnoy D. A. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. J Cell Biol. 1989 Oct;109(4 Pt 1):1597–1608. doi: 10.1083/jcb.109.4.1597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tärnvik A. Nature of protective immunity to Francisella tularensis. Rev Infect Dis. 1989 May-Jun;11(3):440–451. [PubMed] [Google Scholar]
  28. Une T. Studies on the pathogenicity of Yersinia enterocolitica. I. Experimental infection in rabbits. Microbiol Immunol. 1977;21(7):341–363. [PubMed] [Google Scholar]
  29. Wilson C. B., Tsai V., Remington J. S. Failure to trigger the oxidative metabolic burst by normal macrophages: possible mechanism for survival of intracellular pathogens. J Exp Med. 1980 Feb 1;151(2):328–346. doi: 10.1084/jem.151.2.328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yancey R. J., Breeding S. A., Lankford C. E. Enterochelin (enterobactin): virulence factor for Salmonella typhimurium. Infect Immun. 1979 Apr;24(1):174–180. doi: 10.1128/iai.24.1.174-180.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. van Renswoude J., Bridges K. R., Harford J. B., Klausner R. D. Receptor-mediated endocytosis of transferrin and the uptake of fe in K562 cells: identification of a nonlysosomal acidic compartment. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6186–6190. doi: 10.1073/pnas.79.20.6186. [DOI] [PMC free article] [PubMed] [Google Scholar]