QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 24 July 2007
doi: 10.1242/jcs.006221

This Article Summary Full Text Full Text (PDF) Supplementary Material A correction has been published 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Deghmane, A.-E. Articles by Hmama, Z. Search for Related Content PubMed PubMed Citation Articles by Deghmane, A.-E. Articles by Hmama, Z. Social Bookmarking                         What's this?

Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation

¹ Division of Infectious Diseases, Department of Medicine, University of British Columbia and Vancouver Costal Health Institute, Vancouver, British Columbia, V5Z 3J5, Canada
² School of Science and Engineering, Al Akhawayn University, PO Box 104, HII Ave, Ifrane 53 000, Morocco
³ Department of Biochemisty, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan
⁴ Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada

View larger version (40K): [in this window] [in a new window]   Fig. 1. Pathogenic mycobacteria express a 50 kDa coronin-1-interacting protein. (A) Macrophages were exposed to radiolabeled live (L) or gentamycin-killed (GK) BCG or live M. smegmatis (L-Msmeg) for 1 hour at 37°C. Partially attached, non-ingested bacteria were removed by a 5 minute treatment with trypsin-EDTA and extensive washing with HBSS and cells were replenished with culture medium and cultured at 37°C. At 4 hours post-phagocytosis cell lysates were prepared and soluble proteins were mixed with anti-coronin-1 mAb (N-7) or normal mouse IgG (Irr). (B) Cell lysates were prepared as described in A, and then mixed with increasing amounts of purified GST-coronin-1 prior to the addition of N-7 mAb. (C) 35S-CFPs from M. smegmatis, BCG and Mtb were incubated with the soluble fraction of J774 lysates and then mixed with N-7 mAb. Material attached to the N-7 mAb was then immunoprecipitated with protein A-agarose and protein complexes were subjected to SDS-PAGE and autoradiography as described in Materials and Methods (A-C). To ensure that coronin-1 levels remained equivalent at the end of immunoprecipitation process, during the last wash of the immunoprecipitates, 10% from each treatment sample was collected and analyzed by SDS-PAGE and immunoblotting with rabbit polyclonal anti-coronin-1 and the secondary goat anti-rabbit IgG (lower panel). (D) Pulled-down CIP50 from BCG 35S-CFPs was solubilized in 2D-gel rehydration solution, and analyzed by 2D-gel electrophoresis and autoradiography.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Cholesterol stabilizes the binding of CIP50 to coronin-1. (A) Aliquots of 35S-CFPs were mixed with agarose-immobilized GST-tagged coronin-1 (or GST alone), in the absence or in the presence of native or denatured coronin-1-depleted soluble J774 proteins (cell lysate), or in the presence of 1 µM cholesterol (water soluble MCD-cholesterol complex) or 10 µM GM1 or 10 mM MCD. The mixtures were then incubated for 2 hours at 4°C and the agarose beads were washed extensively. J774 lysates were depleted of coronin-1 by multiple cycles of immunoprecipitation with N-7 mAb. To ensure that GST-coronin-1 levels remained equivalent at the end of immunoprecipitation process, during the last wash of the immunoprecipitates, 5% from each treatment sample was collected and analyzed by SDS-PAGE and immunoblotting with anti-GST mAb and the secondary goat anti-mouse IgG (lower panel). (B) J774 cells were infected with radiolabeled BCG for 1 hour as described in Fig. 1 then 10 mM MCD was added to the culture plate for additional 30 minutes. After extensive washing of the cells with HBSS, the culture medium was replenished with or without 1 µM cholesterol, and the cells cultured at 37°C for an additional 4 hours. Cell lysates were then prepared and subjected to immunoprecipitation with N-7 mAb. Pulled-down complexes shown in A and B were analyzed by SDS-PAGE and autoradiography. Lower panel in B shows coronin-1 western blotting of 10% immunoprecipitate from each treatment sample. Band intensities were determined by densitometry using the ImageJ software (http://rsb.info.nih.gov/ij).

View larger version (21K): [in this window] [in a new window]   Fig. 3 . M. smegmatis overexpressing Mtb LpdC retains coronin-1 on its phagosome and survives better within the macrophage. (A) BCG, M. smegmatis (Msmeg) and recombinant M. smegmatis expressing Mtb LpdC (rMsmeg) grown to lag, log and stationary phase in 7H9-OADC were pelleted and cultured (16-18 hours for BCG and 3-4 hours for M. smegmatis) in modified Dubos medium. CFPs corresponding to 108 CFU were 10% TCA precipitated and the culture pellets were lysed by sonication in 75 mM Tris pH 8.8 plus 4 mM EDTA and 100 mM NaCl. CFPs and soluble fractions of bacterial lysates were resolved by SDS-PAGE followed by immunoblotting with anti-LpdC antibodies. (B) J774 cells adhering to glass coverslips were infected with GFP-BCG, GFP-smegmatis (Msmeg) or recombinant M. smegmatis expressing Mtb LpdC and GFP (rMsmeg). At 4 hours post-phagocytosis, cells were fixed, permeabilized and stained with rabbit anti-coronin-1 as described previously (Sendide et al., 2005a). Samples were analyzed by digital confocal microscopy using an epifluorescence microscope. The yellow signal indicates colocalization of green (bacteria) and red (coronin) fluorescence. (C) Quantification of bacterial phagosomes surrounded with coronin-1 observed in 15-20 cells from three individual experiments. (D) Macrophages were infected with the indicated bacteria and at 1 and 4 hours post-phagocytosis, bacteria were released by treatment with 0.1% Triton X-100 in PBS. Thereafter serial dilutions were prepared in PBS and plated on 7H11 plates in triplicate as described previously (Sendide et al., 2004). The data are presented as mean ± s.d. of CFU counts obtained from two independent experiments.

View larger version (49K): [in this window] [in a new window]   Fig. 4. Intracellular trafficking of CIP50/LpdC and its role in mycobacterial survival. (A) J774 cells were infected with gentamycin killed (GK-), live (L-) BCG, M. smegmatis (Msmeg) or M. smegmatis expressing Mtb LpdC (rMsmeg). At 4 hours post-phagocytosis, cells were fixed, permeabilized and stained with N-7 mAb (anti-coronin-1) and Texas Red-conjugated goat anti-mouse IgG. Cells were also stained with anti-LpdC or normal rabbit serum (L-BCG/Irr) and Alexa Fluor 350-conjugated goat anti-rabbit IgG. The enlarged merge panels (L-BCG and rMsmeg), show colocalization (pink color) of coronin-1 (red) with LpdC (blue) in the phagosomal membrane and secreted LpdC (blue) surrounding the phagosome. In the right panel, the yellow signal indicates colocalization of L-BCG and coronin-1 in cells stained with normal rabbit IgG instead of anti-LpdC. Bar, 10 µm. (B) Quantification of LpdC-coronin-1 colocalization in phagosomes observed in 20-25 cells from two individual experiments. (C) Thin sections of BCG-infected cells were fixed with paraformaldehyde and glutaraldehyde and incubated sequentially with rabbit anti-LpdC IgG and gold-conjugated goat anti-rabbit IgG to visualize LpdC (15 nm gold). EM grids were examined with a Tecnai 12 electron microscope at magnification of 37000x (a), 59000x (b) and 97000x (c). The arrows indicate gold particles localized on the phagosomal membrane and the arrowheads indicate gold particles localized in the cytosol. (D) PKH26-stained cells (107/culture plate) were allowed to ingest mycobacteria and phagosomes were prepared at 4 hours post-phagocytosis. Phagosome preparations were stained with normal rabbit IgG or specific antibodies to coronin-1 or LpdC. Preparations were washed and stained with Alexa Fluor 647-labeled secondary antibodies. After adequate compensations, blue fluorescence was analyzed on double (red and green fluorescent)-positive events, which are green fluorescent bacteria surrounded by a red phagosomal membrane (supplementary material Fig. S3); results are expressed as histograms. In each panel, the left histogram represents phagosomes stained with specific antibodies, and the histogram displaced to the right (red) represents phagosomes stained with normal IgG. Values represent the proportion of phagosomes expressing coronin-1 or LpdC.

View larger version (27K): [in this window] [in a new window]   Fig. 5. IFN disrupts CIP50-coronin-1 interaction. (A) Left panel: control and IFN-stimulated (200 U/ml, 24 hours at 37°C) J774 cells were washed and reincubated in the presence of 50 nM LysoTracker Red DND 99 for 2 hours. After three washes with HBSS, cells were then infected with GFP-BCG and chased for 4 hours. Right panel: control and IFN-treated cells were infected with GFP-BCG and stained for coronin-1 as described in Fig. 1. (B) Quantification of BCG-LysoTracker and BCG-coronin-1 colocalizations in phagosomes observed in 15-20 cells from three individual experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Intracellular expression of LRG-47 dislocates coronin-1 from mycobacterial phagosomes. (A) Lysates from control and IFN-stimulated J774 cells were examined by SDS-PAGE and immunoblotting with anti-LRG-47 antibodies, then membranes were stripped and reprobed with anti-actin antibodies. (B) J774 cells were transfected with GFP or GFP-LRG-47 as described in Materials and Methods. In a first series of experiments (a,b), cells were preloaded with LysoTracker Red and infected with BCG expressing Ebfp (BFP-BCG) then fixed at 4 hours post-phagocytosis. In a second series of experiments (c and d), transfected cells were directly infected with BFP-BCG and stained with coronin-1 at 4 hours post-phagocytosis. In b, the white signal indicates simultaneous colocalization of BCG vacuoles with LysoTraker and LRG-47. In c, the magenta signal indicates colocalization of BCG with coronin-1. The cyan signal in d indicates colocalization of BCG phagosome with LRG-47. Short arrows indicate the position of BCG bacilli. (C) Quantification of BCG-LysoTracker and BCG-coronin-1 colocalizations in phagosomes observed in 15-20 cells from two individual experiments. (D) Upper panel: control, IFN-stimulated, GFP- or GFP-LRG-47-transfected cells were infected with radiolabeled BCG and at 4 hours post-phagocytosis, intracellular CIP50-coronin-1 interaction was examined as described in Fig. 1. The relative amounts of CIP50 co-immunoprecipitated with coronin-1 were determined by radioactive count of small aliquots of immunoprecipitated material. Lower panel: total cell lysate form the same treatments were also examined for expression of coronin-1 by western blotting with anti-coronin-1 antibodies.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter