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This Article Full Text Free Full Text (PDF) Free Supplemental Data All Versions of this Article: fj.08-108712v1 23/2/483    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lippuner, C. Articles by Aebischer, T. Search for Related Content PubMed PubMed Citation Articles by Lippuner, C. Articles by Aebischer, T.

Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice

Christoph Lippuner^*,1, Daniel Paape^*,, Athina Paterou, Janko Brand^*, Melville Richardson, Andrew J. Smith, Kirstin Hoffmann^*, Volker Brinkmann, Clare Blackburn and Toni Aebischer^*,,2

^* Department of Molecular Biology and

Central Microscopy Unit, Max-Planck-Institute for Infection Biology, Berlin, Germany; and

Marie Curie Team Pathogen Habitats, Institute of Immunology and Infection Research, and

Institute for Stem Cell Research, University of Edinburgh, Edinburgh, UK

² Correspondence: Marie Curie Team Pathogen Habitats, Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3JT, UK. E-mail: toni.aebischer{at}ed.ac.uk

The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania-infected phagosomes uniformly pass through a Rab5⁺ stage on their intracellular path to compartments with late endosomal/early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to antileishmanial drugs. Here, we generated a unique gfp-rab5 transgenic mouse model to visualize Rab5 recruitment to early parasite-containing phagosomes in primary host cells. Using real-time fluorescence imaging of phagosomes carrying Leishmania mexicana, we determined that parasite-infested phagosomes follow a uniform sequence of transient Rab5 recruitment. Residence in Rab5⁺ compartments was much shorter compared with phagosomes harboring latex beads. Furthermore, a comparative analysis of parasite life-cycle stages and mutants deficient in lpg1, the gene encoding the enzyme required for synthesis of the dominant surface lipophosphoglycan, indicated that parasite surface ligands and host cell receptors modulate pathogen residence times in Rab5⁺ phagosomes, but, as far as tested, had no significant effect on intracellular L. mexicana survival or replication.—Lippuner, C., Paape, D., Paterou, A., Brand, J., Richardson, M., Smith, A. J., Hoffmann, K., Brinkmann, V., Blackburn, C., Aebischer, T. Real-time imaging of Leishmania mexicana-infected early phagosomes: a study using primary macrophages generated from green fluorescent protein-Rab5 transgenic mice.

Key Words: embryonic stem cells • inducible gene expression • doxycycline • wortmannin • reverse tetracycline transactivator • lipophosphoglycan