Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system - PubMed (original) (raw)

Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system

Isabelle Derré et al. Infect Immun. 2004 May.

Abstract

Legionella pneumophila vacuole biogenesis was analyzed by using a cell-free system. We show that calnexin, Sec22b, and Rab1 are recruited to the vacuole very shortly after bacterial uptake, and we have identified Rab1 as a potential host factor involved in the endoplasmic reticulum recruitment process.

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Figures

FIG. 1.

FIG. 1.

Isolation and integrity of L. pneumophila vacuoles. (A) Schematic representation of the procedure used to isolate L. pneumophila vacuoles. The PNS from infected macrophages was layered on top of a discontinuous sucrose gradient. After centrifugation, the vacuoles were concentrated at the 14 to 50% interface. Bar, 5 μm. (B) Efficiency of staining L. pneumophila in the absence (−perm) or presence (+perm) of permeabilization of the vacuolar membranes. Displayed are the mean and standard error of three samples for each condition. Anti-Lp, anti-L. pneumophila antibodies. Solid bars, wild type; open bars, dotA mutant.

FIG. 2.

FIG. 2.

ER recruitment to L. pneumophila vacuoles occurs within 10 min of infection in a Dot/Icm-dependent fashion. (A) Quantification of L. pneumophila vacuoles associated with calnexin 10 min or 1 h postinfection. Solid bars, wild type (WT); open bars, dotA mutant. (B and C) Enhanced recruitment of calnexin after 1 h of infection. Vacuoles were isolated 10 min (B) or 1 h (C) after incubation with either wild-type or dotA mutant bacteria and probed with anticalnexin antibody. Displayed are L. pneumophila (GFP), anticalnexin probing (calnexin), and the two images merged. Bars, 5 μm.

FIG. 3.

FIG. 3.

Rab1 and Sec22b are recruited to the L. pneumophila vacuole in a Dot/Icm-dependent fashion. (A) Quantification of L. pneumophila vacuoles associated with Rab1 or Sec22b 1 h postinfection. Solid bars, wild type (WT); open bars, dotA mutant. (B and C) Punctate pattern of association of Rab1 and Sec22b with L. pneumophila vacuoles. Vacuoles were isolated 1 h after incubation with either wild-type or dotA mutant bacteria and probed with either anti-Rab1 (B) or anti-Sec22b (C). GFP, _L. pneumophila_-GFP. Merge, L. pneumophila displayed in green and either Rab1 or Sec22b displayed in red. Scale bar, 5 μm. (D) Calnexin, Rab1, and Sec22b are associated with vacuoles containing replicating L. pneumophila. Cell lysates from macrophages incubated 14 h with wild-type L. pneumophila were probed with either anticalnexin or anti-Rab1 or anti-Sec22b antibody in the absence of permeabilization. Merge, L. pneumophila displayed in green and either calnexin, Rab1, or Sec22b displayed in red. Bars, 5 μm.

FIG. 4.

FIG. 4.

Overexpression of the GDP-bound form of Rab1a reduces L. pneumophila replication in COS1 cells. (A) The number of viable bacteria was determined every 24 h postinfection by counting CFU. (B) Effect of overexpression of Rab1a derivatives on L. pneumophila intracellular multiplication. COS1 cells were transiently transfected with constructs allowing expression of GFP, the wild-type form of GFP-Rab1a (Rab1aWT), or the GDP-bound form (Rab1aS25N). The infection was allowed to proceed for 14 h, and the number of bacteria per phagosome was scored. (C) Accumulation of Rab1a around the L. pneumophila vacuole in COS1 cells 14 h postinfection. Displayed are L. pneumophila (left panels), GFP-Rab1a derivatives (middle panels), and the two images merged (right panels). Bar, 10 μm.

References

    1. Berger, K. H., and R. R. Isberg. 1993. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol. Microbiol. 7:7-19. - PubMed
    1. Christie, P. J., and J. P. Vogel. 2000. Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol. 8:354-360. - PMC - PubMed
    1. Conover, G. M., I. Derre, J. P. Vogel, and R. R. Isberg. 2003. The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol. Microbiol. 48:305-321. - PubMed
    1. Fraser, D. W., T. R. Tsai, W. Orenstein, W. E. Parkin, H. J. Beecham, R. G. Sharrar, J. Harris, G. F. Mallison, S. M. Martin, J. E. McDade, C. C. Shepard, and P. S. Brachman. 1977. Legionnaires' disease: description of an epidemic of pneumonia. N. Engl. J. Med. 297:1189-1197. - PubMed
    1. Gagnon, E., S. Duclos, C. Rondeau, E. Chevet, P. H. Cameron, O. Steele-Mortimer, J. Paiement, J. J. Bergeron, and M. Desjardins. 2002. Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110:119-131. - PubMed

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