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doi: 10.1242/10.1242/jcs.00034

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Diverting intracellular trafficking of Salmonella to the lysosome through activation of the late endocytic Rab7 by intracellular delivery of muramyl dipeptide

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India

View larger version (20K): [in a new window]   Fig. 1. MBSA-MDP-mediated killing of Salmonella in vitro and in vivo. (A) J774E macrophages (1x106 cells/well) were treated with the indicated concentrations of MBSA-MDP or free MDP for 12 hours. Treated macrophages were infected with Salmonella typhimurium (1x107) as described in the Materials and Methods, and the infected cells were incubated in the respective drug-containing medium. After incubation for 12 hours at 37°C, the macrophages were lysed in solubilization buffer, and an aliquot of the cell lysates was used to determine the level of viable bacteria they contain (colony-forming units). Results are expressed as an average of three determinations±s.d. (B) To determine the efficacy of MBSA-MDP for treatment of Salmonella infection in vivo, C57B1-6 mice were infected with Salmonella as described in the Materials and Methods on day 0. Subsequently, Salmonella-infected animals received intraperitoneal injections of free MDP or MBSA-MDP (1 µg/mouse of MDP equivalent; 50 µg/kg body weight) or ciprofloxacin (1.4 mg/mouse; 70 mg/kg body weight) daily for four consecutive days. Finally, the splenic load of Salmonella was determined by measuring the colony forming units on day 10 as described in the Materials and Methods. Results are expressed as percentage survival of three independent experiments±s.e. The number of colonies in untreated control mice (2.21x104 CFU±s.e.) was taken as 100%.

View larger version (20K): [in a new window]   Fig. 2. Detection of the content of Rab5 and Rab7 in MBSA-MDP-treated cells. J774E cell monolayers were incubated with free or conjugated MDP (MDP equivalent, 1 µg/ml) for 12 hours at 37°C in RPMI-1640 medium. An equivalent number of cells (80 µg of protein each) was solubilized in SDS buffer, boiled and subjected to SDS-PAGE. Western blot analyses were carried out for the detection of actin, Rab5, Rab7 and Rab6 in untreated, MBSA-MDP- or MDP-treated cells. Proteins were visualized using an appropriate HRP-labelled second antibody using ECL. Results from western blots are representative of three independent preparations.

View larger version (23K): [in a new window]   Fig. 3. Intracellular transport of Salmonella in MBSA-MDP-treated cells. (A) Salmonella-containing early phagosomes were purified from untreated cells as well as from MDP and MBSA-MDP-treated cells as described in the Materials and Methods. Proteins from the respective phagosomes (40 µg each) were separated by SDS-PAGE and transferred to nitrocellulose membranes. After incubation with appropriate dilutions of specific antibodies, the proteins on the membranes were visualized using an appropriate HRP-labelled second antibody using ECL. Results from western blots are representative of three independent preparations. (B) In vitro fusion of early endosomes with Salmonella-containing phagosomes isolated from untreated or MBSA-MDP-treated cells. Fusion between early endosomes with respective Salmonella-containing phagosomes isolated from untreated or MBSA-MDP treated cells was carried out either in the presence of normal cytosol or cytosol prepared from MBSA-MDP-treated cells as indicated. Fusion was measured as described in the Materials and Methods. Maximum fusion between endosomes and phagosomes isolated from untreated control cells was observed at 0.5 mg/ml of normal cytosol concentration, which was normalized to one unit. The results are expressed as relative fusion from three independent experiments±s.d. One unit corresponds to 10.2 ng of HRP activity/mg of protein. (C) Intracellular transport of live Salmonella to the lysosomes in MBSA-MDP-treated cells. To determine the transport of Salmonella to the lysosomes by MBSA-MDP-treated cells, J774E cells (1x106 cells) were treated with MDP in the free or conjugated form for 12 hours at 37°C as described earlier. Subsequently, J774E macrophages were preloaded with avidin-HRP and chased for 90 minutes to label the lysosomes. Cells were pulsed with live or dead biotinylated Salmonella at 37°C for a short period of time (5 minutes) to restrict their entry to the early compartment followed by a chase as described in the Materials and Methods. At indicated times the formation of the bacteria biotin-avidin-HRP complex was measured to determine the transport of the Salmonella to lysosomes. Each point represents the mean±s.d. from three independent experiments and is expressed as relative units of transport to lysosomes.

View larger version (36K): [in a new window]   Fig. 4. Reduced Rab5 and enhanced Rab7 content of MBSA-MDP-treated cells is sufficient to drive lysosomal targeting. To determine the role of Rab5 and Rab7 content of MBSA-MDP-treated cells in lysosomal targeting of Salmonella, reconstitution of the phagosome-lysosomes transport assay was carried out in permeabilized cells in the presence of cytosol prepared from MBSA-MDP-treated cells or untreated cells containing in vitro prenylated Rab proteins as described in the Materials and Methods. Cells were incubated for 60 minutes at 37°C to allow transport. Subsequently, formation of the bacteria biotin-avidin-HRP complex was measured to determine the transport of the Salmonella to lysosomes. Each point represents the mean±s.d. from three independent experiments, and these are expressed as relative units of transport to lysosomes.

View larger version (36K): [in a new window]   Fig. 5. MBSA-MDP induced the transport of Salmonella to the lysosomes. (A) To determine the nature of mature LSP, LSP were purified from untreated cells as well as from MBSA-MDP-treated cells as described in the Materials and Methods after 90 minutes of internalization in J774E cells. LSP were washed and the content of actin, cathepsin D and V-ATPase on respective LSP (40 µg each) was determined by western blot analysis using specific antibodies. Results from western blots are representative of three independent preparations. (B) Confocal images showing the GFP-Salmonella colocalization with Lysotracker-Red-labelled lysosomes in untreated (a-c) and MBSA-MDP-treated J774E cells (d-f). Untreated or MBSA-MDP-treated J774E cells were infected with GFP-Salmonella typhimurium and chased for 90 minutes to determine their colocalization with Lysotracker-Red-labelled lysosomes as described in the Materials and Methods. Lysotracker-Red-labelled lysosomes appear in the red channel (a,d); green channel shows the GFP-Salmonella (b,e); and yellow indicates the colocalization of GFP-Salmonella with Lysotracker-labelled lysosomes in merged images (c,f).

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