1887

Abstract

The fungal pathogen has a predilection for the central nervous system (CNS), resulting in devastating meningoencephalitis. At present, it is unclear how traverses the blood–brain barrier (BBB) and causes CNS infection. The present study has examined and characterized the interaction of with human brain microvascular endothelial cells (HBMEC), which constitute the BBB. Adhesion of and transcytosis of HBMEC by was inoculum- and time-dependent and occurred with both encapsulated and acapsulated strains. induced marked morphological changes in HBMEC, for example membrane ruffling, irregular nuclear morphology and swelling of the mitochondria and the ER. These findings suggest that induced actin cytoskeletal reorganization of the host cells. In addition, it was observed that the dephosphorylated form of cofilin was increased during cryptococcal adherence to HBMEC, concomitant with the actin rearrangement. Cryptococcal binding to HBMEC was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of the ROCK←LIMK←cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of to HBMEC, concomitant with the increase of phosphorylation of cofilin. Furthermore, the tight junction marker protein occludin became Triton-extractable, indicating alteration of tight junctions in brain endothelial cells. This is the first demonstration that is able to adhere to and transcytose across the HBMEC monolayer and alter the cytoskeleton morphology in HBMEC. Further characterization of the interactions between and HBMEC should help the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.

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2003-11-01
2024-03-28
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References

  1. Arber S, Barbayannis F. A, Hanser H, Schneider C, Stanyon C. A, Bernard O, Caroni P. 1998; Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393:805–809 [CrossRef]
    [Google Scholar]
  2. Badger J. L, Stins M. F, Kim K. S. 1999; Citrobacter freundii invades and replicates in human brain microvascular endothelial cells. Infect Immun 67:4208–4215
    [Google Scholar]
  3. Bamburg J. R. 1999; Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu Rev Cell Dev Biol 15:185–230 [CrossRef]
    [Google Scholar]
  4. Bar-Sagi D, Feramisco J. R. 1986; Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. Science 233:1061–1068 [CrossRef]
    [Google Scholar]
  5. Bierne H, Gouin E, Roux P, Caroni P, Yin H. L, Cossart P. 2001; A role for cofilin and LIM kinase in Listeria -induced phagocytosis. J Cell Biol 155:101–112 [CrossRef]
    [Google Scholar]
  6. Broadwell R. D, Baker-Cairns B. J, Friden P. M, Oliver C, Villegas J. C. 1996; Transcytosis of protein through the mammalian cerebral epithelium and endothelium.III. Receptor-mediated transcytosis through the blood-brain barrier of blood-borne transferrin and antibody against the transferrin receptor. Exp Neurol 142:47–65 [CrossRef]
    [Google Scholar]
  7. Buchanan K. L, Murphy J. W. 1998; What makes Cryptococcus neoformans a pathogen?. Emerg Infect Dis 4:71–83 [CrossRef]
    [Google Scholar]
  8. Chang Y. C, Kwon-Chung K. J. 1994; Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol 14:4912–4919
    [Google Scholar]
  9. Chang Y. C, Kwon-Chung K. J. 1998; Isolation of the third capsule-associated gene, CAP60 , required for virulence in Cryptococcus neoformans . Infect Immun 66:2230–2236
    [Google Scholar]
  10. Chang Y. C, Kwon-Chung K. J. 1999; Isolation, characterization, and localization of a capsule-associated gene, CAP10 , of Cryptococcus neoformans . J Bacteriol 181:5636–5643
    [Google Scholar]
  11. Chang Y. C, Penoyer L. A, Kwon-Chung K. J. 1996; The second capsule gene of Cryptococcus neoformans , CAP64 , is essential for virulence. Infect Immun 64:1977–1983
    [Google Scholar]
  12. Chen Y. H, Chen S. H, Jong A, Zhou Z. Y, Li W, Suzuki K, Huang S. H. 2002; Enhanced Escherichia coli invasion of human brain microvascular endothelial cells is associated with alternations in cytoskeleton induced by nicotine. Cell Microbiol 4:503–514 [CrossRef]
    [Google Scholar]
  13. Dan C, Kelly A, Bernard O, Minden A. 2001; Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin. J Biol Chem 276:32115–32121 [CrossRef]
    [Google Scholar]
  14. Edwards D. C, Sanders L. C, Bokoch G. M, Gill G. N. 1999; Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat Cell Biol 1:253–259 [CrossRef]
    [Google Scholar]
  15. Eugene E, Hoffmann I, Pujol C, Couraud P. O, Bourdoulous S, Nassif X. 2002; Microvilli-like structures are associated with the internalization of virulent capsulated Neisseria meningitidis into vascular endothelial cells. J Cell Sci 115:1231–1241
    [Google Scholar]
  16. Feldmesser M, Kress Y, Novikoff P, Casadevall A. 2000; Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect Immun 68:4225–4237 [CrossRef]
    [Google Scholar]
  17. Francis C. L, Ryan T. A, Jones B. D, Smith S. J, Falkow S. 1993; Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria. Nature 364:639–642 [CrossRef]
    [Google Scholar]
  18. Galan J. E, Zhou D. 2000; Striking a balance: modulation of the actin cytoskeleton by Salmonella . Proc Natl Acad Sci U S A 97:8754–8761 [CrossRef]
    [Google Scholar]
  19. Gottfredsson M, Perfect J. R. 2000; Fungal meningitis. Semin Neurol 20:307–322 [CrossRef]
    [Google Scholar]
  20. Hamilton A. J, Goodley J. 1996; Virulence factors of Cryptococcus neoformans . Curr Top Med Mycol 7:19–42
    [Google Scholar]
  21. Hirase T, Staddon J. M, Saitou M, Ando-Akatsuka Y, Itoh M, Furuse M, Fujimoto K, Tsukita S, Rubin L. L. 1997; Occludin as a possible determinant of tight junction permeability in endothelial cells. J Cell Sci 110:1603–1613
    [Google Scholar]
  22. Hirase T, Kawashima S, Wong E. Y, Ueyama T, Rikitake Y, Tsukita S, Yokoyama M, Staddon J. M. 2001; Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. J Biol Chem 276:10423–10431 [CrossRef]
    [Google Scholar]
  23. Hogan L. H, Klein B. S, Levitz S. M. 1996; Virulence factors of medically important fungi. Clin Microbiol Rev 9:469–488
    [Google Scholar]
  24. Huang S. H, Jong A. Y. 2001; Cellular mechanisms of microbial proteins contributing to invasion of the blood-brain barrier. Cell Microbiol 3:277–287 [CrossRef]
    [Google Scholar]
  25. Huang S. H, Wass C, Fu Q, Prasadarao N. V, Stins M, Kim K. S. 1995; Escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe10. Infect Immun 63:4470–4475
    [Google Scholar]
  26. Huang S. H, Wan Z. S, Chen Y. H, Jong A. Y, Kim K. S. 2001; Further characterization of Escherichia coli brain microvascular endothelial cell invasion gene ibeA by deletion, complementation, and protein expression. J Infect Dis 183:1071–1078 [CrossRef]
    [Google Scholar]
  27. Ibrahim A. S, Filler S. G, Alcouloumre M. S, Kozel T. R, Edwards J. E Jr, Ghannoum M. A. 1995; Adherence to and damage of endothelial cells by Cryptococcus neoformans in vitro: role of the capsule. Infect Immun 63:4368–4374
    [Google Scholar]
  28. Jong A. Y, Stins M. F, Huang S. H, Chen S. H, Kim K. S. 2001; Traversal of Candida albicans across human blood-brain barrier in vitro. Infect Immun 69:4536–4544 [CrossRef]
    [Google Scholar]
  29. Khan N. A, Wang Y, Kim K. J, Chung J. W, Wass C. A, Kim K. S. 2002; Cytotoxic necrotizing factor-1 contributes to Escherichia coli K1 invasion of the central nervous system. J Biol Chem 277:15607–15612 [CrossRef]
    [Google Scholar]
  30. Kim K. S. 2001; Escherichia coli translocation at the blood-brain barrier. Infect Immun 69:5217–5222 [CrossRef]
    [Google Scholar]
  31. Kuwabara H, Kokai Y, Kojima T, Takakuwa R, Mori M, Sawada N. 2001; Occludin regulates actin cytoskeleton in endothelial cells. Cell Struct Funct 26:109–116 [CrossRef]
    [Google Scholar]
  32. Kwon-Chung K. J, Rhodes J. C. 1986; Encapsulation and melanin formation as indicators of virulence in Cryptococcus neoformans . Infect Immun 51:218–223
    [Google Scholar]
  33. Kwon-Chung K. J, Sorrell T. C, Dromer F, Fung E, Levitz S. M. 2000; Cryptococcosis: clinical and biological aspects. Med Mycol 38:Suppl. 1205–213 [CrossRef]
    [Google Scholar]
  34. Maekawa M, Ishizaki T, Boku S. 7 other authors 1999; Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285:895–898 [CrossRef]
    [Google Scholar]
  35. Masuda M, Betancourt L, Matsuzawa T, Kashimoto T, Takao T, Shimonishi Y, Horiguchi Y. 2000; Activation of rho through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin. EMBO J 19:521–530 [CrossRef]
    [Google Scholar]
  36. Matter K, Balda M. S. 1999; Occludin and the functions of tight junctions. Int Rev Cytol 186:117–146
    [Google Scholar]
  37. Mitchell T. G, Perfect J. R. 1995; Cryptococcosis in the era of AIDS – 100 years after the discovery of Cryptococcus neoformans . Clin Microbiol Rev 8:515–548
    [Google Scholar]
  38. Nakano K, Takaishi K, Kodama A, Mammoto A, Shiozaki H, Monden M, Takai Y. 1999; Distinct actions and cooperative roles of ROCK and mDia in Rho small G protein-induced reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells. Mol Biol Cell 10:2481–2491 [CrossRef]
    [Google Scholar]
  39. Niwa R, Nagata-Ohashi K, Takeichi M, Mizuno K, Uemura T. 2002; Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108:233–246 [CrossRef]
    [Google Scholar]
  40. Nizet V, Kim K. S, Stins M, Jonas M, Chi E. Y, Nguyen D, Rubens C. E. 1997; Invasion of brain microvascular endothelial cells by group B streptococci. Infect Immun 65:5074–5081
    [Google Scholar]
  41. Nunoue K, Ohashi K, Okano I, Mizuno K. 1995; LIMK-1 and LIMK-2, two members of a LIM motif-containing protein kinase family. Oncogene 11:701–710
    [Google Scholar]
  42. Perfect J. R, Wong B, Chang Y. C, Kwon-Chung K. J, Williamson P. R. 1998; Cryptococcus neoformans : virulence and host defences. Med Mycol 36:Suppl. 179–86
    [Google Scholar]
  43. Rubin L. L, Staddon J. M. 1999; The cell biology of the blood-brain barrier. Annu Rev Neurosci 22:11–28 [CrossRef]
    [Google Scholar]
  44. Sears C. L. 2000; Molecular physiology and pathophysiology of tight junctions.V. Assault of the tight junction by enteric pathogens. Am J Physiol Gastrointest Liver Physiol 279:G1129–G1134
    [Google Scholar]
  45. Simonovic I, Arpin M, Koutsouris A, Falk-Krzesinski H. J, Hecht G. 2001; Enteropathogenic Escherichia coli activates ezrin, which participates in disruption of tight junction barrier function. Infect Immun 69:5679–5688 [CrossRef]
    [Google Scholar]
  46. Song Y, Hoang B. Q, Chang D. D. 2002; ROCK-II-induced membrane blebbing and chromatin condensation require actin cytoskeleton. Exp Cell Res 278:45–52 [CrossRef]
    [Google Scholar]
  47. Stins M. F, Gilles F, Kim K. S. 1997a; Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 76:81–90 [CrossRef]
    [Google Scholar]
  48. Stins M. F, Prasadarao N. V, Zhou J, Arditi M, Kim K. S. 1997b; Bovine brain microvascular endothelial cells transfected with SV40-large T antigen: development of an immortalized cell line to study pathophysiology of CNS disease. In Vitro Cell Dev Biol Anim 33:243–247 [CrossRef]
    [Google Scholar]
  49. Sumi T, Matsumoto K, Takai Y, Nakamura T. 1999; Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2. J Cell Biol 147:1519–1532 [CrossRef]
    [Google Scholar]
  50. Sumi T, Matsumoto K, Nakamura T. 2001; Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase. J Biol Chem 276:670–676 [CrossRef]
    [Google Scholar]
  51. Tsukita S, Furuse M, Itoh M. 1999; Structural and signalling molecules come together at tight junctions. Curr Opin Cell Biol 11:628–633 [CrossRef]
    [Google Scholar]
  52. Wachtel M, Frei K, Ehler E, Fontana A, Winterhalter K, Gloor S. M. 1999; Occludin proteolysis and increased permeability in endothelial cells through tyrosine phosphatase inhibition. J Cell Sci 112:4347–4356
    [Google Scholar]
  53. Wyrick P. B, Choong J, Davis C. H, Knight S. T, Royal M. O, Maslow A. S, Bagnell C. R. 1989; Entry of genital Chlamydia trachomatis into polarized human epithelial cells. Infect Immun 57:2378–2389
    [Google Scholar]
  54. Yang N, Higuchi O, Ohashi K, Nagata K, Wada A, Kangawa K, Nishida E, Mizuno K. 1998; Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393:809–812 [CrossRef]
    [Google Scholar]
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