Characterization of Mycobacterium bovis Phagosome L. Jordao * , M. Simoes * , C. Bleck ** , G. Griffiths ** and E. Anes * * CPM-URIA, Faculty of Pharmacy, University of Lisbon, Avenida das Forcas Armadas, 1600-085- Lisbon, Portugal, ** EMBL, Meyerhofstraße 1, 69117 Heidelberg, Germany eanes@ff.ul.pt Mycobacterium tuberculosis complex are among the most successful pathogens. Their success resides in the ability to interfere with intracellular traffic avoiding natural pathways of the phagosome maturation [1]. Recently, mycobacteria escape from the phagosome to the cytosol was investigated as an alternative survival strategy. In this context we decided to determine the exact intracellular location of Mycobacterium bovis in different host macrophages and characterize the pathogen intracellular niche for survival. The main goal here was to characterize live vs dead M. bovis spp phagosome in different host macrophages. Macrophages infection, fluorescence and EM procedures were carried out as described previously [2-4]. Although it is accepted that intracellular mycobacteria reside inside the phagosome, a few studies showed that they are able to escape and persist in the cytosol [5-7]. Our data is in agreement with the persistence inside a phagosome (Fig.1(a)). The lack of acidification is a clear indicator of phagosome maturation arrest. The data presented in table.1 shows that live BCG and M. bovis can arrest phagosome acidification, thus being independently of the host used. In contrast phagosome containing heat-killed mycobacteria fully matures within 3 days. When we evaluated phagolysosome biogenesis following either the fusion with colloidal gold particles (targeted to lysosomes) or the acquisition of the v-ATPase (responsible for pH drop within the phagosome lumen) (Fig.1(b)) the same conclusion is achieved. Figure 1(c) shows that live and heat-killed BCG resides in different compartments in J774 cells. While less than 20 % of phagosomes containing live BCG co-localize with late lysosome markers (LAMP-1, LYAAT and LBPA) the phagosome containing heat-killed BCG acquired most of these markers and much faster. The rate of EEA1 acquisition also supports this assumption. All together our data clearly show that live mycobacteria persist within an immature membrane limited compartment. Acknowledgements This work was financed by a grant and a PhD fellow from FCT (POCI/BIA-BCM/55327/2004; SFRH/BD/14284/2003). We are grateful to Antonio Pedro Matos, Michael Niederweis and Helena Ferronha for their support and/ or mycobacteria strains. References [1] D. G. Russell , Nat. Rev. Mol. Cell Biol. 2 (2001) 569 [2] L. Jordao et al., Cell Microbiol. (2007) [3] E. Anes et al., Nat. Cell Biol. 5 (2003) 793 [4] E. Anes et al., Cell Microbiol. 8 (2006) 939 [5] K. A. McDonough et al., Infect. Immun. 61 (1993) 2763 [6] Q. N. Myrvik et al., Am. Rev. Respir. Dis. 129 (1984) 322 [7] N van der Wel et al. , Cell 129 (2007) 1287 Microsc Microanal 14 (supp 3), 2008 124 doi: 10.1017/S1431927608089629 Copyright 2008, LASPM