Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1994 Nov;62(11):5126–5132. doi: 10.1128/iai.62.11.5126-5132.1994

Inhibition of infection of macrophages with Ehrlichia risticii by cytochalasins, monodansylcadaverine, and taxol.

Y Rikihisa 1, Y Zhang 1, J Park 1
PMCID: PMC303234  PMID: 7927796

Abstract

The requirement of functions of clathrin, microfilaments, and microtubules in binding, internalization, proliferation, and spreading of Ehrlichia risticii in macrophages was studied. Monodansylcadaverine (MDC), which inhibits clustering and internalization of the ligand-receptor complexes into clathrin-coated vesicles; cytochalasin B or D, which depolymerizes microfilaments; and taxol, which binds and stabilizes polymerized microtubules, were found to prevent ehrlichial infection in murine peritoneal macrophages when they were present throughout the infection period. [35S]methionine-labeled ehrlichial binding to the macrophage was reduced by 0 to 22%; therefore, the binding was not the major point of inhibition. However, MDC, cytochalasin D, and taxol inhibited ehrlichial internalization into macrophages by 80, 58, and 32%, respectively. When MDC, cytochalasin B or D, or taxol was added immediately after internalization of E. risticii (3 h postinfection), ehrlichial replication in P388D1 cells was almost completely prevented. Also, all of these agents almost completely prevented ehrlichial spreading from P388D1 cells to THP-1 human monocytes. These agents were not found to be ehrlichiacidal when approximately 40%-infected P388D1 cells were treated for 2 days, although further intracellular proliferation was prevented. Furthermore, none of these compounds directly inhibited the metabolic activity of E. risticii, since 14CO2 production from L-[14C]glutamine by Percoll density gradient-purified host-cell-free E. risticii was not shown to be impaired. The action of taxol was probably due to impairment of the microtubule function since a microtubule-depolymerizing agent, colchicine, also inhibited intracellular proliferation of E. risticii. Neither reactive oxygen intermediates, nitric oxide, nor tumor necrosis factor alpha appeared to be involved in taxol-induced inhibition of E. risticii in macrophages. Thus, our findings indicate that ehrlichial internalization appears to take place by a mechanism that is more dependent on the functions of clathrin and less dependent on the functions of microfilaments or microtubules. Replication within macrophages and intercellular spreading appear to require clathrin, microfilaments, and microtubules. Consequently, alteration of these structures with inhibitors can result in complete prevention of infection.

Full text

PDF
5126

Selected References

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

  1. Aggeler J., Werb Z. Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin. J Cell Biol. 1982 Sep;94(3):613–623. doi: 10.1083/jcb.94.3.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axline S. G., Reaven E. P. Inhibition of phagocytosis and plasma membrane mobility of the cultivated macrophage by cytochalasin B. Role of subplasmalemmal microfilaments. J Cell Biol. 1974 Sep;62(3):647–659. doi: 10.1083/jcb.62.3.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cooper J. A. Effects of cytochalasin and phalloidin on actin. J Cell Biol. 1987 Oct;105(4):1473–1478. doi: 10.1083/jcb.105.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Desiderio J. V., Kiener P. A., Lin P. F., Warr G. A. Protection of mice against Listeria monocytogenes infection by recombinant human tumor necrosis factor alpha. Infect Immun. 1989 May;57(5):1615–1617. doi: 10.1128/iai.57.5.1615-1617.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ding A. H., Nathan C. F., Stuehr D. J. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol. 1988 Oct 1;141(7):2407–2412. [PubMed] [Google Scholar]
  6. Ding A. H., Porteu F., Sanchez E., Nathan C. F. Shared actions of endotoxin and taxol on TNF receptors and TNF release. Science. 1990 Apr 20;248(4953):370–372. doi: 10.1126/science.1970196. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Green L. C., Wagner D. A., Glogowski J., Skipper P. L., Wishnok J. S., Tannenbaum S. R. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982 Oct;126(1):131–138. doi: 10.1016/0003-2697(82)90118-x. [DOI] [PubMed] [Google Scholar]
  9. Hodinka R. L., Davis C. H., Choong J., Wyrick P. B. Ultrastructural study of endocytosis of Chlamydia trachomatis by McCoy cells. Infect Immun. 1988 Jun;56(6):1456–1463. doi: 10.1128/iai.56.6.1456-1463.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hodinka R. L., Wyrick P. B. Ultrastructural study of mode of entry of Chlamydia psittaci into L-929 cells. Infect Immun. 1986 Dec;54(3):855–863. doi: 10.1128/iai.54.3.855-863.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Khan M. N., Khan R. J., Posner B. I. A method for quantitation of protein in the presence of Percoll. Anal Biochem. 1981 Oct;117(1):108–112. doi: 10.1016/0003-2697(81)90699-0. [DOI] [PubMed] [Google Scholar]
  12. Leung M. F., Sokoloski J. A., Sartorelli A. C. Changes in microtubules, microtubule-associated proteins, and intermediate filaments during the differentiation of HL-60 leukemia cells. Cancer Res. 1992 Feb 15;52(4):949–954. [PubMed] [Google Scholar]
  13. Levitzki A., Willingham M., Pastan I. Evidence for participation of transglutaminase in receptor-mediated endocytosis. Proc Natl Acad Sci U S A. 1980 May;77(5):2706–2710. doi: 10.1073/pnas.77.5.2706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Majeed M., Kihlström E. Mobilization of F-actin and clathrin during redistribution of Chlamydia trachomatis to an intracellular site in eucaryotic cells. Infect Immun. 1991 Dec;59(12):4465–4472. doi: 10.1128/iai.59.12.4465-4472.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Margolis R. L., Wilson L. Addition of colchicine--tubulin complex to microtubule ends: the mechanism of substoichiometric colchicine poisoning. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3466–3470. doi: 10.1073/pnas.74.8.3466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Marsh M., Helenius A. Adsorptive endocytosis of Semliki Forest virus. J Mol Biol. 1980 Sep 25;142(3):439–454. doi: 10.1016/0022-2836(80)90281-8. [DOI] [PubMed] [Google Scholar]
  17. Matteoni R., Kreis T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol. 1987 Sep;105(3):1253–1265. doi: 10.1083/jcb.105.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Messick J. B., Rikihisa Y. Characterization of Ehrlichia risticii binding, internalization, and proliferation in host cells by flow cytometry. Infect Immun. 1993 Sep;61(9):3803–3810. doi: 10.1128/iai.61.9.3803-3810.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Murray H. W., Cohn Z. A. Macrophage oxygen-dependent antimicrobial activity. I. Susceptibility of Toxoplasma gondii to oxygen intermediates. J Exp Med. 1979 Oct 1;150(4):938–949. doi: 10.1084/jem.150.4.938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Newman S. L., Mikus L. K., Tucci M. A. Differential requirements for cellular cytoskeleton in human macrophage complement receptor- and Fc receptor-mediated phagocytosis. J Immunol. 1991 Feb 1;146(3):967–974. [PubMed] [Google Scholar]
  22. Park J., Rikihisa Y. Inhibition of Ehrlichia risticii infection in murine peritoneal macrophages by gamma interferon, a calcium ionophore, and concanavalin A. Infect Immun. 1991 Oct;59(10):3418–3423. doi: 10.1128/iai.59.10.3418-3423.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Park J., Rikihisa Y. L-arginine-dependent killing of intracellular Ehrlichia risticii by macrophages treated with gamma interferon. Infect Immun. 1992 Sep;60(9):3504–3508. doi: 10.1128/iai.60.9.3504-3508.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rikihisa Y. Growth of Ehrlichia risticii in human colonic epithelial cells. Ann N Y Acad Sci. 1990;590:104–110. doi: 10.1111/j.1749-6632.1990.tb42212.x. [DOI] [PubMed] [Google Scholar]
  25. Rikihisa Y., Ito S. Entry of Rickettsia tsutsugamushi into polymorphonuclear leukocytes. Infect Immun. 1982 Oct;38(1):343–350. doi: 10.1128/iai.38.1.343-350.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rikihisa Y. The tribe Ehrlichieae and ehrlichial diseases. Clin Microbiol Rev. 1991 Jul;4(3):286–308. doi: 10.1128/cmr.4.3.286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salisbury J. L., Condeelis J. S., Satir P. Role of coated vesicles, microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B lymphoblastoid cells. J Cell Biol. 1980 Oct;87(1):132–141. doi: 10.1083/jcb.87.1.132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sandvig K., van Deurs B. Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis. J Biol Chem. 1990 Apr 15;265(11):6382–6388. [PubMed] [Google Scholar]
  29. Schiff P. B., Fant J., Horwitz S. B. Promotion of microtubule assembly in vitro by taxol. Nature. 1979 Feb 22;277(5698):665–667. doi: 10.1038/277665a0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Shemer-Avni Y., Wallach D., Sarov I. Inhibition of Chlamydia trachomatis growth by recombinant tumor necrosis factor. Infect Immun. 1988 Sep;56(9):2503–2506. doi: 10.1128/iai.56.9.2503-2506.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Talbot P. J., Vance D. E. Biochemical studies on the entry of sindbis virus into BHK-21 cells and the effect of NH4Cl. Virology. 1982 Apr 30;118(2):451–455. doi: 10.1016/0042-6822(82)90365-8. [DOI] [PubMed] [Google Scholar]
  33. Walker T. S., Winkler H. H. Penetration of cultured mouse fibroblasts (L cells) by Rickettsia prowazeki. Infect Immun. 1978 Oct;22(1):200–208. doi: 10.1128/iai.22.1.200-208.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wall D. A., Wilson G., Hubbard A. L. The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes. Cell. 1980 Aug;21(1):79–93. doi: 10.1016/0092-8674(80)90116-6. [DOI] [PubMed] [Google Scholar]
  35. Wani M. C., Taylor H. L., Wall M. E., Coggon P., McPhail A. T. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc. 1971 May 5;93(9):2325–2327. doi: 10.1021/ja00738a045. [DOI] [PubMed] [Google Scholar]
  36. Ward M. E., Murray A. Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: mechanisms of endocytosis. J Gen Microbiol. 1984 Jul;130(7):1765–1780. doi: 10.1099/00221287-130-7-1765. [DOI] [PubMed] [Google Scholar]
  37. Weiss E., Dasch G. A., Kang Y. H., Westfall H. N. Substrate utilization by Ehrlichia sennetsu and Ehrlichia risticii separated from host constituents by renografin gradient centrifugation. J Bacteriol. 1988 Nov;170(11):5012–5017. doi: 10.1128/jb.170.11.5012-5017.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weiss E., Williams J. C., Dasch G. A., Kang Y. H. Energy metabolism of monocytic Ehrlichia. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1674–1678. doi: 10.1073/pnas.86.5.1674. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES