Summary
Phagocytosis is a process by which invading organisms are taken up by macrophages and targeted to the lysosomes, where they are degraded. However, many pathogens modulate this central process of macrophage-mediated killing by inhibiting their transport to the lysosomes through a variety of pathogen-derived mechanisms. Given the importance of Rab proteins in the regulation of intracellular transport pathways, we investigated the role of different host endocytic Rabs on the maturation of Salmonella-containing phagosomes in macrophages. Initially, we have developed a ligand mixing assay to measure the transport of the Salmonella-containing phagosomes to lysosomes. Using this assay we have shown that Salmonella decline their transport to the lysosomes. In order to determine whether inhibition of Salmonella transport to lysosomes is due to their sustained fusion with early endosomes, we have developed an in vitro fusion assay between Salmonella-containing phagosomes and early endosomes. Here, we have discussed how these methodologies are helpful to determine the mechanism of evasion of Salmonella transport to the lysosomes.
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References
Garcia-del Portillo, F. (1999) Pathogenic interference with host vacuolar trafficking. Trends Microbiol. 6, 467–469.
Russell, D. G., Xu, S. M., and Chakraborty, P. (1992) Intracellular trafficking and the parasitophorous vacuole of Leishmania mexicana infected macrophages. J. Cell. Sci. 103, 1193–1210.
Portnoy, D. A., Jacks, P. S., and Hinrichs, D. J. (1988) Role of hemolysin for the intracellular growth of Listeria monocytogenes. J. Exp. Med. 167, 1459–1471.
Hall, B. F., Webster, P., Ma, A. K., Joiner, K. A., and Andrew, N. W. (1992) Desialy-lation of lysosomal membrane glycoprotein by Trypanosoma cruzi: a role for the surface neuraminidase in facilitating parasite entry into the host cell cytoplasm. J. Exp. Med. 176, 313–325.
Vergne, I., Chua, J., Lee, H. H., Lucas, M., Belisle, J., and Deretic, V. (2005) Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis Proc. Natl. Acad. Sci. USA 102, 4033–4038.
Rathman, M., Sjaastad, M. D., and Falkow, S. (1996) Acidification of phagosomes containing Salmonella typhimurium in murine macrophages. Infect. Immun. 64, 2765–2773.
Buchmeier, N. A. and Heffron, F. (1991) Inhibition of macrophage phagosome-lysosome fusion by Salmonella typhimurium. Infect. Immun. 59, 2232–2238.
Zerial, M. and McBride, H. (2001) Rab proteins as membrane organizers. Nat. Rev. Mol. Cell. Biol. 2, 107–117.
Mukhopadhyay, A., Barbieri, A. M., Funato, K., Roberts, R. and Stahl, P. D. (1997) Sequential actions of rab5 and rab7 regulate endocytosis in the Xenopus oocyte. J. Cell Biol. 136, 1227–1237.
Gorvel, J. P., Chavrier, P., Zerial, M. and Gruenberg, J. (1991) Rab5 controls early endosome fusion in vitro. Cell 64, 915–925.
Mukhopadhyay, A., Funato, F. and Stahl, P. D. (1997) Rab7 regulates transport from early to late endocytic compartments in Xenopus oocytes. J. Biol. Chem. 272, 13055–13059.
Feng, Y., Press, B. and Wandinger-Ness, A. (1995) Rab7: an important regulator of late endocytic membrane traffic. J. Cell Biol. 131, 1435–1452.
Pitt, A., Mayorga, L. S., Schwartz, A. L., and Stahl, P. D. (1992) Transport of phagosomal components to an endosomal compartment. J. Biol. Chem. 267, 126–132.
Qiu, Y., Xu, X., Wandinger-Ness, A., Dalke, D. P., and Pierce, S. (1994) Separation of subcellular compartments containing distinct functional forms of MHC class II. J. Cell Biol. 125, 595–605.
Mukherjee, K., Siddiqi, S. A., Hashim, S., Raje, M., Basu, S. K. and Mukhopadhyay, A. (2000) Live Salmonella recruits N-ethylmaleimide–sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome J. Cell. Biol. 148, 741–754.
Funato, F., Baron, W., Yang, C. Z., Mukhopadhyay, A. and Stahl, P. D. (1997) Reconstitution of phagosome-lysosome fusion in streptolysin-permeabilized cells. J. Biol. Chem. 272, 16147–16151.
Hashim, S., Mukherjee, K., Raje, M., Basu, S. K. and Mukhopadhyay, A. (2000) Live Salmonella modulate expression of rab proteins to persist in a specialized compartment and escape transport to lysosomes. J. Biol. Chem. 275, 16281–16288.
Colombo, M. I., Taddese, M., Whiteheart, S. W., and Stahl, P. D. (1996) A possible predocking attachment site for N-ethylmaleimide-sensitive fusion protein. Insight from in vitro endosome fusion. J. Biol. Chem. 271, 18810–18816.
Chen, L. M., Kaniga, K., and Galan, J.E. (1996) Salmonella spp. are cytotoxic for cultured macrophages. Mol. Microbiol. 21, 1101–1115.
Acknowledgements
This work was supported by grants from the department of Biotechnology and Indian Council of Medical Research, Government of India. R. M. is supported by a research fellowship from the Council of Scientific and Industrial Research and G. K. is supported by a research fellowship from National Institute of Immunology, India.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Madan, R., Krishnamurthy, G., Mukhopadhyay, A. (2008). SopE-Mediated Recruitment of Host Rab5 on Phagosomes Inhibits Salmonella Transport to Lysosomes. In: Deretic, V. (eds) Autophagosome and Phagosome. Methods in Molecular Biology™, vol 445. Humana Press. https://doi.org/10.1007/978-1-59745-157-4_27
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DOI: https://doi.org/10.1007/978-1-59745-157-4_27
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