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97, 435–447, May 14, 1999, Copyright ©1999 by Cell Press
A Coat Protein on Phagosomes Involved in the Intracellular Survival of Mycobacteria
Giorgio Ferrari,* Hanno Langen,† Makoto Naito,‡ and Jean Pieters*§ * Basel Institute for Immunology Grenzacherstrasse 487 CH-4005 Basel Switzerland † Hoffmann-La Roche CH-4002 Basel Switzerland ‡ Department of Pathology Niigata University School of Medicine Niigata 951-8510 Japan events along the phagosomal and endocytic pathway to lysosomes, where such material is degraded (Desjardins et al., 1994; Russell, 1995). In the case of mycobacteria, early observations suggested that phagosomes containing living mycobacteria resist fusion with organelles of the endosomal/lysosomal system (Armstrong and D’Arcy Hart, 1971). This inhibition depends on active processes exerted by the mycobacteria, as dead mycobacteria are readily transported to endosomal/lysosomal organelles (Armstrong and D’Arcy Hart, 1971; Barker et al., 1997; Hasan et al., 1997). This resistance of the mycobacterial phagosome to fusion with endosomal/ lysosomal organelles is thought to be related to the ability of mycobacteria to escape from macrophage bactericidal mechanisms (Small et al., 1994; Russell, 1995). Morphological characterization of the mycobacterial phagosome showed that this organelle contains markers of the early but lacks markers of later stages of the endocytic pathway, such as late endosomes and lysosomes (Sturgill-Koszycki et al., 1994; Clemens and Horwitz, 1995; Barker et al., 1997). Interestingly, the mycobacterial phagosome selectively excludes the proton-ATPase that is normally responsible for acidification along the endocytic pathway (Mellman et al., 1985; Sturgill-Koszycki et al., 1994). The resulting limited acidification of the mycobacterial phagosome is thought to represent a key mechanism allowing mycobacterial survival. However, the molecular mechanisms by which the mycobacterial phagosome resists fusion with or maturation into lysosomes remain largely unknown. As an approach toward the identification of components involved in these processes, we have developed a system in which phagosomes containing living mycobacteria could be physically separated from phagosomes containing dead bacilli. We reasoned that biochemical analysis of these two populations of vesicles might allow the identification of factors that contribute to the intraphagosomal survival of mycobacteria. We here report the identification and characterization of a WD repeat–containing molecule, termed TACO (for tryptophane aspartate–containing coat protein), that was associated with phagosomes containing live, but not with those containing killed, bacteria. TACO represents a coat protein that transiently associated with phagosomes but was actively retained on mycobacterial phagosomes as long as the bacteria were viable. The capacity of viable mycobacteria to retain TACO on the phagosomal membrane, thereby preventing their delivery to lysosomes, thus represents a mechanism by which these pathogens are able to escape host defense mechanisms.
Summary Mycobacteria are intracellular pathogens that can survive within macrophage phagosomes, thereby evading host defense strategies by largely unknown mechanisms. We have identified a WD repeat host protein that was recruited to and actively retained on phagosomes by living, but not dead, mycobacteria. This protein, termed TACO, represents a component of the phagosome coat that is normally released prior to phagosome fusion with or maturation into lysosomes. In macrophages lacking TACO, mycobacteria were readily transported to lysosomes followed by their degradation. Expression of TACO in nonmacrophages prevented lysosomal delivery of mycobacteria and prolonged their intracellular survival. Active retention of TACO on phagosomes by living mycobacteria thus represents a mechanism preventing cargo delivery to lysosomes, allowing mycobacteria to survive within macrophages.
Introduction Pathogenicity of microorganisms is often related to their ability to survive and replicate within their mammalian hosts. After entry, a pathogen should find a suitable site for survival and replication within the cell while at the same time evade or circumvent host defense mechanisms. As many pathogens have coevolved with the organisms that they infect, they may use host cellular components for their entry, survival, and growth (Falkow, 1991; Bliska et al., 1993; Beverley, 1996; Galan and Bliska, 1996; Finlay and Cossart, 1997). One example of a successful exploitation of cellular mechanisms to survive is provided by the interaction of Mycobacterium species with macrophages. Whereas the normal function of macrophages is to engulf and destroy microorganisms, mycobacteria have evolved ways to circumvent the hostile environment of the macrophage (Russell, 1995). Phagocytosed material is normally transported via a series of fission and fusion
§ To whom correspondence should be addressed (e-mail: pieters@
Results Biochemical Characterization of Mycobacterial Phagosomes To study the organelles in which mycobacteria reside inside macrophages, the interaction of M. bovis BCG
lower panel). From infected macrophages (upper panels. dead). To analyze the molecular composition of mycobacteria-containing phagosomes. as analyzed by the presence of bacteria (Figure 1a. To separate the different subcellular compartments. “Live”. left. To analyze the protein composition of the phagosomes containing viable as well as those containing heat-killed mycobacteria. right. after which a homogenate was prepared. together with late endosomal/lysosomal membranes (Figure 1b. bovis BCG. The major spot in phagosomes containing viable mycobacteria represents actin. to prevent excessive degradation of the bacilli) resulted in their recovery in shifted fractions. “Dead”. Organelles were subsequently separated by organelle electrophoresis. left. bacteria containing fractions after organelle electrophoresis were pooled and phagosomes were prepared by low-speed centrifugation as described in Experimental Procedures. followed by a 12 (live) or 2 (dead) hr chase. using this method. right. Detection of a 50 kDa Protein Present in Phagosomes Containing Viable Mycobacteria (a and b) Subcellular fractionation of phagosomes containing viable (a) and heat-killed (b) mycobacteria. basic. lower panel). a late endosomal/lysosomal .. Thus. Separately. organelle electrophoresis was used (Tulp et al. from uninfected macrophages a late endosomal/lysosomal fraction as well as a lysosomal fraction (from pooling exclusively the most anodally -hexosaminidase containing shifted membranes) was prepared by organelle electrophoresis and high-speed centrifugation (lower panels). The murine macrophage cell line J774 was infected for 1 hr with M. Macrophages that had been metabolically labeled with 35S-methionine/cysteine were infected with live BCG for 1 hr. subcellular fractionation was utilized. whereas mycobacterial phagosomes remain in unshifted fractions. the bacteria-containing fractions were pooled and sedimented at low speed to prepare a phagosomal fraction as described in Experimental Procedures. late endosomes and lysosomes shift toward the anode (Figures 1a and 1b. From each fraction. Acidic. vacuoles containing live mycobacteria could be physically separated from those containing dead mycobacteria. with the murine macrophage cell line J774A. live. and chased for 12 hr prior to homogenization and subcellular fractionation. The 50 kDa protein specifically associated with phagosomes containing live bacteria is indicated by an arrow. (c) Analysis of proteins present in subcellular fractions isolated from metabolically labeled macrophages.Cell 436 Figure 1. Killing of the mycobacteria by heat treatment prior to uptake by macrophages for 1 hr followed by a 2 hr chase (instead of 12 hr.1 was investigated. During such a separation procedure. Proteins from phagosomes were separated by two-dimensional IEF/SDS–PAGE and analyzed by fluorography. and the distribution of the organelle-specific markers as indicated were determined (upper panels). 1994). Separately. upper panels). washed. the amount of mycobacteria was quantitated by direct observation by light microscopy after acid fast staining (lower panels).
segment bordered by Gly-His (GH) and Trp-Asp (WD) dipeptide residues. The gel was coelectrophoresed with different organelle fractions to allow alignment of the phagosomal 50 kDa spot (arrow). Most polypeptides present in phagosomes containing living mycobacteria (Figure 1c. several additional molecules of the same molecular weight but differing in isoelectric point were detected on the fluorograph. arrow) that was not detected in any of the other subcellular fractions analyzed. membrane fractions were subjected to two-dimensional IEF/SDS–PAGE. WD repeats are characterized by a 30–40 amino acid residue Figure 2. 1994). To confirm that this reading frame encoded the 50 kDa protein that accumulated in mycobacterial phagosomes. Shown is the spectrum obtained after mass spectrometry. all of which were present in lysosomes prepared from an uninfected homogenate (Figure 1c. Based on this homology. Cloning and Sequencing of the Phagosomal Protein of 50 kDa To identify the 50 kDa protein. matching amino acid residues 11–14 (Figure 2b). as well as from metabolically labeled bacteria (data not shown). a lys-C digest of the polypeptide isolated from the twodimensional gel was separated by HPLC. indicating that it represented a host protein. “Lysosomes”). 1995). Furthermore. Identification of the 50 kDa Protein (a) The polyacrylamide gel piece containing the 50 kDa protein (see Figure 1) was excised and subjected to lys-C digestion and mass spectrometry. The 50 kDa polypeptide was absent from fluorographs prepared from a similar subcellular fraction in the absence of infection. possibly representing post-translationally modified forms or. a 50 kDa polypeptide was present (Figure 1c. the cDNA encoding the mouse homolog of this protein was cloned from a macrophage library as described in Experimental Procedures. a human WD repeat protein of unknown function. The five WD repeats present in the sequence are underlined. A BLAST search with the 50 kDa protein sequence revealed homology to IR10 (40% identity). termed coronin (35% identity). in phagosomal membranes containing live mycobacteria.. Sequencing of positive clones revealed an open reading frame of 1386 base pairs encoding a 461 amino acid residue polypeptide with a predicted sequence shown in Figure 2b. including phagosomes containing killed mycobacteria (Figure 1c. antibodies raised against the C-terminal amino acid sequence were used to immunoprecipitate the 50 kDa protein from lysates of noninfected macrophages that had been metabolically labeled. as well as to a WD repeat protein identified in Dictyostelium discoideum. “Late endosomes/Lysosomes”). Nine out of 11 peptides matched the digestion profile of entry P31146 (SwissProt).Mycobacterial Subversion of Phagocytosis 437 fraction as well as a lysosomal fraction was prepared from uninfected macrophages by organelle electrophoresis and high-speed sedimentation. Phagosomes containing killed bacteria were enriched for a subset of lysosomal proteins (compare Figure 1c. Sequence analysis of the peptide eluting at 21 min by Edman degradation revealed the sequence FRHV. (b) Predicted amino acid sequence of the 50 kDa protein.. alternatively. “Phagosomes (Dead)”). representing a 57 kDa protein previously identified in human cells (Suzuki et al. Interestingly. Analysis by two-dimensional IEF/SDS–PAGE and coelectrophoresis with phagosomal organelles showed that the immunoprecipitated protein migrated at the identical position as the phagosomal 50 kDa protein (Figure 2c). In addition. However. and after Coomassie blue staining the 50 kDa polypeptide was excised and subjected to lys-C protease digestion and mass spectrometry (Figure 2a). an actinbinding protein involved in actin-based cytoskeletal rearrangements. right hand panels). analysis of the primary sequence using Prosite revealed the lack of a signal sequence for targeting to the endoplasmic reticulum as . and could be detected in total homogenates from uninfected macrophages. The primary sequence of the 50 kDa polypeptide contains five WD repeats (underlined in Figure 2b). but their function remains unknown (Neer et al. “Phagosomes (Live)”) could also be detected in endosomal/lysosomal organelles from uninfected macrophages (Figure 1c. closely related proteins. Protein patterns from these organelles were analyzed by two-dimensional IEF/SDS–PAGE followed by fluorography (Figure 1c). (c) Immunoprecipitation using anti-TACO antibodies of cell lysates from metabolically labeled macrophages followed by two-dimensional IEF/SDS–PAGE.
After stripping. When heat-killed bacteria were allowed to be internalized by macrophages. permeabilized. later). a rat monoclonal antibody raised against TACO predominantly labeled the cortex of the cells and colocalized with tubulin. (Lower left) Localization of actin (Texas red) and tubulin (FITC). we have named this protein TACO for tryptophane aspartate– containing coat protein. The different tissues as indicated were isolated from a mouse. Localization of TACO in Infected Cells Since TACO was identified as a component of phagosomes containing viable mycobacteria. TACO dissociated from phagosomes containing killed bacilli within the first 2 hr and redistributed to the cell cortex during this time period (Figure 4c). TACO strongly colocalized with tubulin (Figure 3). Double labeling of cells for actin and tubulin showed only a partial colocalization (Figure 3b). Subcellular Localization of TACO The subcellular distribution of TACO was analyzed morphologically using confocal immunofluorescence microscopy.. consistent with a function in macrophages and macrophage-like cells. homogenized. In contrast. TACO is expressed in cells of the lymphoid/myeloid lineage. To analyze the tissue distribution of TACO. with the notable exception of Kupffer cells (see later). middle panel) as well as for tubulin (Figure 3a. Thus. proteins from a homogenate prepared from various tissues were separated by SDS– PAGE and transferred to nitrocellulose (Figure 3a).Cell 438 well as the presence of a coiled-coil region within the 32 C-terminal amino acid residues (Berger et al. as well as a small amount in lung. cortex over the next few hours. thus carrying out functions that are essential for normal cellular growth and therefore expected to be expressed in all cell types (Neer et al. the immunoblot was stripped and stained for actin (Figure 3a. and brain. In noninfected macrophages. and stained for tubulin (upper left) followed by Texas red–labeled secondary antibodies or F-actin using phalloidin-Texas red (upper right) and double labeled for TACO using a rat monoclonal antibody followed by FITC-labeled secondary antibodies. but not with actin (Figure 3b). Because of the presence of the WD repeats as well as its morphological characterization (cf. fixed. 1994). washed and trypsinized to remove adherent bacteria. lower panel). Macrophages were grown on coverslips. To visualize mycobacteria-containing phagosomes. but not in any other tissue examined (Figure 3a. TACO became almost completely relocalized to the phagosomal membrane and remained associated with the phagosome up to prolonged periods of time (Figure 4a). and as tubulin has been implicated in phagosome–lysosome fusion (Desjardins . Cells were allowed to internalize mycobacteria for 1 hr. Together these results suggest that in noninfected macrophages. immunoreactivity was found to be present in spleen. (Lower right) Inclusion of the immunizing peptide blocked TACO staining. in addition to a cortical localization. bone marrow–derived and peritoneal macrophages as well as all macrophage cell lines analyzed expressed TACO (data not shown). lymph nodes. Distribution and Localization of TACO (a) Tissue distribution of TACO. Tissue Distribution of TACO Several members of the WD repeat family are involved in regulating cytoskeletal assembly and membrane traffic. upper panel). As a control. TACO accumulated initially at the site of bacterial uptake but redistributed to the cell Figure 3. Immediately after infection (chase 0 hr). In addition.. 5 m. Quantitation of TACO localization at the phagosome revealed that 80%–90% of the mycobacterial phagosomes stained positive for TACO in case of viable bacilli at all time points. and total proteins (15 g) separated by SDS–PAGE and immunoblotted using anti-TACO antiserum (upper panel). indicating the presence of two distinct cortical cytoskeletal elements in these cells. Bar. After staining of the membrane using anti-TACO antibodies. TACO is associated with the cortical microtubule network. Within the first 2 hr. TACO was localized around the mycobacterial phagosomes. bovis BCG expressing green fluorescent protein (BCG-GFP) was utilized. resulting in a staining pattern as in noninfected cells (Figure 4b). the subcellular distribution in cells infected with mycobacteria was analyzed. thymus. and cultured at 37 C for the times indicated in Figure 4a. M. In noninfected macrophages. 1995). (b) Subcellular localization of TACO in uninfected cells. the blot was reincubated with anti-actin antibodies (middle panel) or anti-tubulin antibodies (lower panel).
After fixation and permeabilization. bovis BCG expressing the green fluorescent protein (BCG-GFP) for 1 hr followed by a chase for the times indicated. cells were scored for the presence of TACO at the mycobacterial phagosome. Subcellular Localization of TACO in Infected Cells Macrophages were grown on coverslips and infected with live (a) or heat-killed (b) M.Mycobacterial Subversion of Phagocytosis 439 Figure 4. cells were infected with live (left) or heat-killed (right) mycobacteria followed by fixation and staining using antitubulin antibodies and Texas red–labeled secondary antibodies. Shown are mean values ( SD) from three experiments (n 50). the cells were stained for TACO using mAb GK3 followed by Texas red–labeled secondary antibodies. In (d). (c) For quantitation. For each field. a bright field image is shown (left side). 5 m. . Bar. Bar. 5 m.
1994) or. As shown in Figure 4d.. Effect of Rifampicin and Alkalinization on TACO Distribution (a) Cells were infected for 1 hr with live mycobacteria. lower panels). alternatively. consistent with a role for microtubules in phagosome–lysosome fusion (Desjardins et al. despite the presence of large numbers of macrophages. cells infected with live bacteria were incubated for 2 hr in medium containing the anti-mycobacterial drug rifampicin. To directly analyze phagocytosis of mycobacteria in TACO-negative Kupffer cells. virtually all bacilli were retrieved in unshifted fractions. Quantitation (right side) revealed that whereas 90% of the phagosomes containing viable bacilli had recruited TACO. the liver did not show any expression of TACO (Figure 3a). the Kupffer cells (Wardle. 5 m. and stained for TACO as described.and rifampicin-killed mycobacteria might be due to differences in bacterial treatment. virtually none of those containing killed bacilli colocalized with TACO. et al. confirming the ability of NH4Cl to inhibit phagosome–lysosome fusion (data not shown)... 1991. 1994). fixed. (b) Cells were alkalinized as described in Experimental Procedures and subsequently infected with live (upper panels) or heat-killed (lower panels) mycobacteria for 1 hr. 1982. Sturgill-Koszycki et al.Cell 440 Figure 5. Whereas in nontreated cells 90% of the mycobacterial phagosomes contained TACO (Figure 5a.. as expected. followed by a 2 hr chase in the absence (upper panels) or presence (lower panels) of rifampicin (1 mg/ml). Cells . upper panels). lower panels). To distinguish between these possibilities. Blocker et al. phagosomes containing heat-killed mycobacteria formed in the presence of NH4Cl were analyzed. Localization of Mycobacteria in TACO-Negative Kupffer Cells In contrast to all other macrophage-containing tissue. TACO was not retained on phagosomes containing heat-killed bacilli (Figure 5b.. these cells were isolated from the liver and infected with mycobacteria for 1 hr followed by a 2 hr chase. The difference in TACO recruitment between phagosomes containing heat. the distribution of tubulin in macrophages infected with live as well as with heat-killed mycobacteria was analyzed. be dictated by processes exerted by viable bacteria. When organelles from NH4Cl-treated macrophages infected with heat-killed bacteria were separated by organelle electrophoresis. Quantitation of TACO recruitment (as in Figure 4) revealed a strong reduction of TACO colocalization with mycobacteria (right side). To analyze whether the recruitment and retention of TACO required viability of the mycobacteria. After fixation. The absence of TACO from Kupffer cells was confirmed by immunoblotting of a lysate prepared from isolated Kupffer cells as well as by immunohistochemistry on liver sections (Figures 6a and 6c). in cells infected with live mycobacteria tubulin rapidly dissociated from the mycobacterial phagosome. upper panels). Ammonium chloride inhibits phagosome–lysosome fusion (Gordon et al. Besides tubulin. cells were stained for TACO as described. This further indicates that lack of acidification per se is not responsible for TACO retention but rather processes exerted by the mycobacteria themselves. Hart and Young. followed by a 2 hr chase. Kielian and Cohn. the in situ killing of the bacteria by rifampicin resulted in a rapid release of TACO from the phagosomal membrane (Figure 5a. Bar. Retention of TACO on phagosomes may occur as a result of the elevated pH in phagosomes as opposed to lysosomes (Crowle et al. 1989). 1991) without altering the morphology or distribution of phagosomes (Heuser. 1987). 1994. also LAMP was recruited to the phagosomes containing killed bacilli (results not shown). 1980.. 1998). whereas tubulin remained present at all times around phagosomes containing killed bacilli. Immunofluorescence analysis of these cells revealed that while ammonium chloride treatment did not affect TACO recruitment on phagosomes containing living mycobacteria (Figure 5b.
after an overnight chase virtually all bacilli were degraded (Figure 6b. Mice were untreated (c) or infected (d) with M. in most if not all phagocytosing Kupffer cells. Thus. LAMP-TR). (a) Kupffer cells were isolated from liver as described in Experimental Procedures.. 1974). lysosomal vacuoles could be found recruited to and concentrated at the site of bacterial uptake. (c and d) Immunohistochemistry of TACO expression in mouse liver. one consequence of a mycobacterial challenge in vivo can be the formation of liver granulomas. lysed. in macrophages lacking TACO. a J774 lysate (10 g) was used. bovis BCG expressing the green fluorescent protein (BCG-GFP). and no mycobacteria could be detected in LAMP-negative vacuoles. followed by a complete destruction of the bacilli. were subsequently fixed and stained for lysosomes using antibodies against lysosomal-associated membrane glycoprotein (LAMP) and analyzed by confocal immunofluorescence microscopy (Figure 6b. as they stained posi- . upper panels).Mycobacterial Subversion of Phagocytosis 441 Figure 6. bovis BCG. lower panels). TACO-positive macrophages represented exclusively infiltrated macrophages. Immunohistochemical Analysis of TACO Expression Although the liver Kupffer cells are known to completely eradicate bacterial infections (North. the granuloma regions stained positive for TACO. and sections were obtained from the liver. Interestingly. All phagocytosed bacilli were present in lysosomal organelles. sacrificed. 5 m. Bar. whereas after a 2 hr chase phagolysosomes could be detected harboring both intact bacilli as well as partially degraded mycobacteria (as judged from the green fluorescent protein distribution pattern). Cells were washed and incubated for 2 hr (upper panels) or 16 hr (lower panels) prior to fixation and staining for lysosomal glycoprotein followed by Texas red–labeled secondary antibodies (LAMP-TR). and total proteins (10 g) separated by SDS–PAGE followed by immunoblotting using anti-TACO antibodies. TACO Expression and Mycobacterial Entry in Liver Macrophages (a and b) Localization of mycobacteria in TACO-negative Kupffer cells. and these macrophages harbored mycobacteria as revealed by Ziehl-Neelsen staining (Figure 6d). Immunohistochemistry was performed using the rat mAb GK3 followed by HRP-coupled secondary antibodies (left panel). When liver sections obtained from BCGinfected mice were analyzed. (b) Isolated Kupffer cells were incubated for 1 hr with M. As a control. thought to be involved in restricting the bacterial spread (Gordon et al. or double labeling was performed using mAb GK3 and Ziehl-Neelsen staining. Granulomas are macrophage-rich lesions harboring viable bacilli. All of the infected Kupffer cells observed contained large vesicular structures that strongly stained positive for LAMP (Figure 6b. mycobacteria were rapidly taken up in lysosomes. In fact. 1994).
when present. When Mel JuSo cells were infected for 1 hr with mycobacteria followed by a 3 hr chase.. after homogenization and subcellular fractionation by electrophoresis 15% of the internalized bacilli were located in shifted fractions. To directly analyze this. washed. cells were lysed in PBS containing 0.Cell 442 Figure 7. At the chase times indicated. was transfected with cDNA encoding TACO. However. 1989). Differential Mycobacterial Trafficking and Survival in Nonphagocytes Expressing TACO The recruitment of TACO on phagosomes containing live mycobacteria together with the resistance of these mycobacterial phagosomes to fuse with or mature into lysosomes suggests that TACO functions as a component of the phagosomal coat that. transfer of the mycobacteria to late endosomes was completely abrogated. and bacteria were collected by centrifugation and resuspended in 7H9 medium.. Nibbering et al. Serial dilutions were plated out and the number of colonies determined from the appropriate dilution. prevents fusion with lysosomes. whereas no colocalization at all was observed in TACO-transfected cells (data not shown). lower panels). and chased. Shown is a representative experiment out of three. Differential Trafficking and Survival of Mycobacteria in TACO-Transfected Cells Mel JuSo cells transfected with cDNA encoding TACO in the sense (“TACO-transfected”) or antisense (“Control”) orientation were lysed and total proteins (10 g) separated by SDS–PAGE followed by immunoblotting using anti-TACO antibodies (a). In accordance with this. tive both for F4/80 and Mac-I antigens. indicating a limited ability of these cells to target mycobacteria to late endosomal/lysosomal organelles (Figure 7b). which was in fact three orders of magnitude lower when compared to J774 macrophages (Figure 7d and data not shown). in control Mel JuSo cells a subset of intracellularly residing bacteria colocalized with LAMP ( 10%). 1981. Shown are mean values ( SD) from five experiments (d and e). upper panels). and the distribution of the organelle-specific markers as indicated was determined (b and c. (e) TACO-transfected or control cells were incubated with mycobacteria. washed. and subjected to organelle electrophoresis. From each fraction. homogenized. Control (b) or TACO-transfected (c) cells were incubated with mycobacteria for 1 hr followed by a 3 hr chase. and the total uptake was determined by liquid scintillation counting of the lysate. (d) TACO-transfected or control cells were infected with metabolically labeled mycobacteria for 1 hr at 37 C. as shown by the absence of mycobacteria from shifted fractions (Figure 7c). which does not express TACO.5% Triton X-100. and lysed. . the nonphagocytic melanoma cell line Mel JuSo. in TACO-transfected Mel JuSo cells. the former staining all macrophage cell types. and stably transfected cell lines were selected (Figure 7a). Transfection of TACO did not affect mycobacterial uptake. the amount of mycobacteria was analyzed by direct observation by light microscopy after acid fast staining (b and c. the latter being not reactive with Kupffer cells (Hirsch et al.
Expression of TACO in a nonmacrophage cell line was sufficient to prevent lysosomal fusion of the mycobacterial phagosome.. TACO Is a WD Repeat Protein The predicted amino acid sequence of TACO showed that it is a member of the WD repeat protein family (Neer et al.. Strikingly. which is thought to represent a key mechanism by which mycobacteria manage to survive intracellularly. also in nonphagocytic but TACO-expressing cells. washed. while it dissociates from the microtubule network. Clemens and Horwitz. cells were lysed and the amount of viable mycobacteria determined by counting the formation of colonies after plating out different dilutions on agar. 1998). Blocker et al. and it is conceivable that during the 1500–2000 million years that separate Dictyostelium from mammals (Knoll.. Although the precise function of the WD repeat remains unknown. de Hostos et al. one function of this protein is associated with particle uptake (Maniak et al. However. Interestingly. 1997)... Most studies to date have implicated the actin cytoskeleton in phagocytosis of a wide variety of living and inert particles (Greenberg et al. TACO initially associates with phagosomes but is rapidly released concomitant with lysosomal delivery of the bacilli. 1992). TACO shows 35% homology to a WD repeat. motility (King et al. 1995). a protein associated with phagosomes containing viable but not killed mycobacteria. WD repeat–containing proteins are involved in a variety of processes. 1997). cytoskeletal organization. Thus. in TACO-negative Kupffer cells all bacilli were located in lysosomes followed by their degradation. these cells were infected with mycobacteria for 1 hr. tubulin remained associated exclusively with phagosomes containing killed bacteria. Both the actin and tubulin cytoskeleton may play a role during the initial phase of bacterial internalization to generate the forces required for the actual internalization process. C phosphorylation consensus sites present in TACO are possibly involved in such regulation.. including signal transduction. Together these results suggest that TACO is actively retained on mycobacterial phagosomes by processes exerted by the mycobacteria themselves and is one of the factors providing a niche for mycobacteria to survive within macrophages. and further incubated for the times indicated in Figure 7e. 1995). 1995). In agreement with these reports. 1995).. 1998). possibly as a result of its initial association with microtubules (black lines) at the cell cortex. 1995. Cellular slime molds such as Dictyostelium live on soil bacteria. Members of this family are present throughout eukaryotic evolution and thus far have not been found to be present in prokaryotic organisms (Neer et al. the almost complete colocalization of TACO with tubulin in noninfected macrophages suggests that at least some part of TACO either directly or indirectly binds to microtubules. including mycobacteria. termed coronin (de Hostos et al. Moreover. otherwise distinct WD repeat proteins in processes requiring microtubule-based activity (Li and Suprenant.Mycobacterial Subversion of Phagocytosis 443 To analyze mycobacterial survival in TACO-transfected versus control Mel JuSo cells. This indicates that in this organism. 1995). one of which leads to a reduced capacity to internalize yeast particles (Maniak et al. tubulin rapidly dissociated from TACOcoated phagosomes containing viable mycobacteria. Suzuki et al. 1993). Russell. At each time point. 1993). In contrast to actin. As a result. Discussion A hallmark of the mycobacterial phagosome is its resistance to fusion with other stages of the endosomal/ lysosomal pathway (Armstrong and D’Arcy Hart. Salama et al. cytokinesis (Shirayama et al. 1994.. and the predicted protein kinase Figure 8. 1994). we found that phagosomes containing viable as well as those containing dead bacilli were associated with actin. Given the role of several. 1994.. we have identified TACO. 1990. Rathman et al. TACO is actively retained on the phagosomal membrane... 1994. in TACO-transfected Mel JuSo cells survival of mycobacteria was 3-fold higher within the first 3 hr after infection and increased to 8-fold after an overnight chase (Figure 7e). 1971. In a search for host cell components that are involved in the intracellular survival of mycobacteria.. Wilkerson et al. 1991. actin-binding protein identified in the slime mold Dictyostelium discoideum. 1993. mycobacteria have acquired the ability to actively recruit . after the internalization of living mycobacteria. In contrast. during phagocytosis of killed bacteria (black) or after in situ killing of the bacilli. and vesicle fusion (Pryer et al.. 1995. Model Describing the Role of TACO in Intracellular Survival of Mycobacteria Phagocytosis triggers the recruitment of TACO (yellow) around the particle to be ingested.. consistent with previous reports showing that fusion of phagosomes with lysosomes is a microtubule-dependent process (Desjardins et al. In the case of viable bacteria (violet). Deletion of the gene encoding this protein in Dictyostelium results in an impaired functioning of actin-based processes. delivery of mycobacteria to lysosomes is prevented. allowing survival of mycobacteria. Microtubule binding might be regulated by posttranslational modifications. it is tempting to speculate that the WD repeat represents a microtubule-binding domain.. thereby prolonging their survival. perhaps as a consequence of a conformational change. Retention of TACO on the phagosomal membrane was strictly dependent on mycobacterial viability and not due to impaired acidification... Rauchenberger et al. mycobacteria have the capacity to resist their delivery to late endosomal/lysosomal organelles.
Japan). 1991)... Cosson and Letourneur. another WD repeat protein. Mice (BALB/c) were obtained from IffaCredo (Basel. It is precisely at this last step in the normal maturation pathway of phagocytosed material that mycobacteria intervene. 1997). and copurification may have resulted from the colocalization of both proteins at the cortical cytoskeleton (Woodman et al. Defining the binding partners of TACO at the phagosomal membrane as well as in the cytosol might identify the complexes involved in such signal transduction processes. (Tokyo.Cell 444 and retain host cell components. Mice were sacrificed and the thoracic cavity exposed followed by canulation of the inferior vena cava via the right atrium. For example. 1978. The resulting cell suspension was filtered through dressing sponges (Johnson and Johnson. release of TACO would be required to trigger lysosomal fusion. Switzerland) or from Charles River. 4 . Interestingly. this indicates that TACO is not absolutely essential for phagocytosis. 1997). 1996). Clearance of bacteria in the liver is attributed to the presence of large amounts of Kupffer cells (North. 1995. Sathyamoorthy et al. After 15 min. 1998).. three types of coats have been defined to function in distinct steps of vesicular traffic. the liver was perfused with 10 ml of PBS followed by perfusion of a collagenase solution (5 mg/ml collagenase D [Boehringer Mannheim] in RPMI-1640. TACO might in fact prevent the budding and fusion machinery to be recruited onto the plasma membrane and therefore identify this membrane as nonfusogenic with lysosomes. these results suggest that at the level of the whole organism rather than at the single cell. Schekman and Orci. evolutionary pressure may have ensured downregulation of TACO expression in Kupffer cells to provide an efficient and complete mycobacterial clearance site. 1996. might have a very similar scaffolding function for lysosomes (Perou et al. 1981). 1981) was grown in RPMI-1640 containing 5% heatinactivated fetal calf serum. Indeed. Why have mammalian cells retained a molecule that allows mycobacteria to survive within macrophages? One possibility is that TACO performs another.. 1996). including TACO.. as the processes involved in cell locomotion and phagocytosis are quite similar (Berlin et al. In human neutrophiles. Arlington. The liver was removed and mechanically disrupted prior to incubation with 10 ml of 1 mg/ ml of the same collagenase solution at 37 C. Cells were centrifuged five times (30 g. Once material is being phagocytosed. 1996. the Kupffer cells. when isolated Kupffer cells were infected with mycobacteria. First.. one function of the WD repeat proteins might be to provide a scaffold for the different organelles to maintain their integrity.. Thus far. As transfection of TACO in nonphagocytic cells also inhibits mycobacterial delivery to late endosomes. while COPI. Kupffer cells were isolated according to Lee et al. However. Vesicular coat proteins are transiently recruited onto membranes and required in a defined budding or fusion step or during organelle maturation to transfer cargo from one stage to another (Rothman and Wieland. mycobacteria have gained the capacity to actively retain TACO on the phagosomal membrane. The human melanoma cell line Mel JuSo (Johnson et al. TX). have made use of the normal cellular processes involved in their own uptake to circumvent host defense responses is yet another striking example of the ingenuity of pathogenic microorganisms. the contribution of p40 in the NADPH oxidase is unclear (Tsunawaki et al. functioning both in the invagination of large pieces of plasma membrane. and the filtrate was diluted to 50 ml in RPMI containing 10% heat-inactivated FCS.. (1986) with the following modifications. rather than being delivered to lysosomes for degradation (see also Figure 8). in the course of their coevolution with higher eukaryotes. as yet unknown function in macrophages. the beige/ lyst gene product. thereby possibly preventing the recruitment of the machinery necessary for cargo delivery to lysosomes (Figure 8). Clathrin-based coats function in cargo delivery to the endocytic pathway as well as to post-Golgi organelles (Kirchhausen et al. Recruitment and retention of TACO on the phagosomal membrane might be regulated through transmembrane signal transduction processes induced by the living bacteria. DNAse (Sigma) from a 5 mg/ml stock solution in PBS was added to a final concentration of 0. that ensure their survival.. The fact that mycobacteria.. did not. In fact. Experimental Procedures Cells.. The liver is the prime target for infectious agents that have accessed an organism. After opening of the portal vein to allow efflux. 1997). prewarmed at 37 C). 1990).5 mg/ml. and cells lacking an intact beige/lyst product are characterized by the presence of abnormally large lysosomes whose function is impaired (Burkhardt et al. Bacteria. The homology of TACO with Dictyostelium coronin would indeed argue in favor of such a role. but as an organ it is rarely infected (Brandborg and Goldman. Inc. as well as in forming protrusions of the plasma membrane involved in cell locomotion (Valerius et al. a TACO homolog was found to copurify with the p40 protein thought to play a role in the NADPH oxidase (Grogan et al. 1981). Valerius et al. all of the bacilli were rapidly transferred to lysosomes and degraded. To avoid lysosomal delivery.. Also this protein colocalizes with microtubules (Faigle et al. this function of TACO appears to be independent from the phagocytic machinery with which macrophages are equipped. TACO may be a crucial component of the cytoskeleton of highly motile cells.(for coat protein I) and COPII-based coats are involved in traffic between the endoplasmic reticulum and Golgi organelles (Bednarek et al. Retention of TACO by Mycobacteria to Prevent Lysosomal Delivery The distribution of TACO during phagocytosis suggests that it represents a coat protein of the mycobacterial phagosome. Second. 1974. and it is possible that phagosomal transmembrane proteins are involved in the regulation of TACO retention. and Antibodies The murine macrophage cell line J774A. How mycobacteria achieve this remains to be established. 1987). Instead of functioning in cargo selection and/or budding processes. whereas the resident liver macrophages. Wardle..1 was obtained from ATCC and cultured in DMEM containing 10% heat-inactivated fetal calf serum at 37 C in 5% CO2. 1993). A Balance between the Host and the Pathogen Evidence for a role for TACO in vivo in the containment of mycobacteria in a viable state was provided by the finding that infiltrated macrophages in liver granulomas expressed TACO. 1997).
One microliter from the separated liquid was applied onto the dried matrix spot. The resulting cell population consisted of 90% macrophages as revealed by F4/80 staining. For mass spectrometry. 1991). Immunoprecipitations were performed as described (Pieters et al. cDNA Cloning and Sequencing To obtain the cDNA encoding TACO. mouse IgG1) and -tubulin (mouse IgG1) were obtained from Boehringer Mannheim and Amersham.. The digestion was performed for 3–12 hr at room temperature.. and a mass tolerance of 0.1 M ammonium bicarbonate and washed with water. Ferrari et al. After incubation. 1994.8). Tokyo. cDNAs were introduced into Mel JuSo cells by electroporation using the BioRad Gene Pulser (400 V/125 F). dissolved in physiological saline at a concentration of 10 mg/ml. San Diego CA). and antibodies were affinity purified by affinity chromatography using the immunizing peptides as ligands. For the construction of a eukaryotic expression construct. cells were fixed in methanol (4 min at 20 C). Coverslips were incubated with primary antibodies for 40 min at room temperature. Protein Analysis and Mass Spectrometry Proteins were analyzed using one-dimensional (10%) SDS–PAGE according to Laemmli (1970) or two-dimensional IEF/SDS–PAGE as described (Lefkovits et al. bovis BCG (Japan BCG Inc.. and phagosomes were collected by low-speed centrifugation (30 min. and clones were selected using G418. Killing of mycobacteria was carried out by heat treatment for 30 min at 80 C.1% trifluoroacetic acid was added. washed in PBS. containing 1 g of endopeptidase Lys-C (Wako) after drying in a speedvac evaporator.5 l) was applied on the sample target. 1997).. Briefly. centrifuged for 2 min. CA). digested in situ. CA) attached to an Axiovert 35M microscope (Carl Zeiss. The liver was removed at various intervals after BCG injection and processed for immunohistochemistry. Leiden). Monoclonal antibodies were generated by immunizing rats (Wistar) with the same peptides and fusion of the spleen and lymph nodes with SP2/0 myeloma cells. Inc. Thornwood. Japan). Calibration was internal to the samples with des-Arg-1 Bradykinin (Sigma) and adrenocorticotropic hormone fragment 18-39 (Sigma). Subcellular Fractionation and Phagosome Isolation Cells were homogenized and organelles prepared essentially as described (Tulp et al.Mycobacterial Subversion of Phagocytosis 445 min) at room temperature. respectively. The protein spot of interest was excised. For immunohistochemistry. Quantitation of the total mycobacterial uptake was performed by incubating cells for 1 hr with 35S-methionine/cysteine-labeled mycobacteria. Leiden. and a representative field is shown. Immunofluorescence Microscopy and Immunohistochemistry Cells were grown on glass coverslips and infected as described above. and the rat mAb F4/80 (IgG2b) was kindly provided by Dr. Hercules. twodimensional gels were fixed and stained using colloidal Coomassie (Novex. cells were plated out. bovis BCG harboring phsp60gfp (BCG-GFP) (Dhandayuthapani et al. a TACO-encoding fragment was subcloned into pBL-SK after digestion of TACOpBL-SK with XhoI and XbaI.000 g. UK). Sequencing was performed using the ABI PRISM Dye terminator cycle sequencing reaction (Perkin Elmer. 1995) was kindly provided by Dr. Mycobacterium bovis BCG. and the infection was performed as described above. The gel pieces were reswollen with 5 l of 3 mM Tris-HCl (pH 8... Deretic (University of Michigan. Birmingham. and permeabilized in 0. After 3 weeks. Hasan et al. Alkalinization was performed as described in Heuser (1989). In vivo infection was performed by injecting 8-week-old BALB/c mice intravenously with 0. Mycobacterial survival was analyzed by lysing cells in D-PBS containing 0. 1997). The membrane pellet was resuspended in homogenization buffer. NY). 1985). Stratagene) was screened with a probe amplified from human spleen cDNA (Clonetech) using the following primers: 5 -CCAGTGCTATGAAGATGTGCGCG-3 (forward primer) and 5 -ATGATGCGCACGCGCTTGTCACGGC-3 (reverse primer).15 mCi/ml 35S-methionine/cysteine mix (Promix. excised spots were destained with 30% acetonitrile in 0. a mouse macrophage ZAP II library (from peritoneal macrophages. and then incubated with FITC and Texas red coupled goat anti-rabbit and/or goat anti-mouse antibodies (Southern Biotechnology Associates. cells were lysed in Dulbecco’s phosphate-buffered saline (D-PBS) containing 1% Triton X-100 and analyzed by liquid scintillation counting. Labeling was performed by placing the cells in methionine/cysteine-free medium containing 10% dialyzed FCS and 0. For the protein search. Coverslips were mounted in FluoroGuard Antifade Mounting Reagent (Bio Rad) and analyzed using a confocal laser scanning microscope system (MRC-600. 1997. washed in PBS containing 2% BSA. MI) and propagated in 7H9 mycobacterial medium (Difco) supplemented with 10% OADC Middlebrook supplement (Difco). For each condition as well as for quantitation. S.1%) was included in all subsequent incubations... Alternatively. the number of colonies formed was determined from the appropriate dilutions.5% Triton X-100 and sedimentation of the bacteria at 2700 g for 10 min. 150 bacteria-containing cells were analyzed. Foster City. Matrix solution (0. and characterized by matrix-assisted laser desorption ionization mass spectrometry (Fountoulakis and Langen. August and obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. After extensive washing. Saponin (0. After two final washes in RPMI-FCS. cells were washed three times in Ringer’s solution without NH4Cl. The pellet was resuspended in 7H9 medium. and organelle electrophoresis as well as the determination of organelle specific markers was carried out as described (Ferrari et al. A TACO-encoding fragment was generated using EcoRI and ligated into the mammalian expression vector pUC-CAGGS (Niiwa et al. Three microliters of 30% acetonitrile containing 0. and different dilutions were plated out on 7H11 agar (Difco). Amersham). In short. The presence of mycobacteria was determined by centrifugation of the fractions onto glass coverslips followed by acid fast staining (Difco) and quantitation by direct observation of the coverslips. Phagosomes were isolated after pooling of the bacteria-containing fractions followed by sedimentation at 100. strain Montreal was kindly provided by Dr. after which the nonadherent cells were removed by five washes in RPMI. Polyclonal antisera were raised against KLH-coupled peptides in New Zealand white rabbits.1% saponin in PBS for 30 min at room temperature. Metabolic Labeling and Immunoprecipitation Prior to labeling. Ann Arbor. 1997). macrophages were grown for 30 min in methionine/ cysteine-free medium containing 10% dialyzed FCS. Thole (TNO. The rat anti-murine LAMP-1 antibody 1D4B (IgG2a) was developed by T. Antibodies against actin (clone C4.. and the content was vortexed. Actin was visualized using phalloidin-Texas red (Molecular Probes. 1991) either in the antisense or sense orientation.. and M. The Netherlands). Bio Rad laboratories. 380 g). An acceleration voltage of 20 kV was used. left to adhere for 60 min at 37 C. 1997). monoisotopic peptide masses were used..006% was allowed. followed by equilibration of the cells (two times for 15 min each) in Ringer’s solution containing 30 mM NH4Cl (alkalinization medium). and the purified fragment ligated into pBK-CMV (Stratagene) digested with the same enzymes. The matrix consisted of 15 mg of nitrocellulose (Bio-Rad) and 20 mg of -cyano 4-hydroxycinnamic acid (Sigma) dissolved in 1 ml of acetone isopropanol 1:1 (v/v). The membrane pellet was resuspended in 6% Ficoll-70 in homogenization buffer. and sonicated for 5 min. CA) and analyzed using an ABI 373 DNA sequencer. the liver was fixed for 4 hr at 4 C in . AL). and the pBluescript phagemid containing the cloned DNA inserts was excised by coinfection with helper phage in XL1-Blue MRF to yield TACOpBL-SK. Bacteria were washed in the same alkalinization medium. Rat anti-Mac-1 antibody (IgG2b) was from CALTAG (San Francisco. supplemented with 10% OADC Middlebrook and glycerol (5 ml/l). cells were fixed in 3% paraformaldehyde (at 37 C) for 10 min.2 ml of M. Gordon (Oxford. The samples were analyzed in a time-of-flight Perseptive Biosystems mass spectrometer (Voyager Elite) equipped with a reflector and delayed extraction. Infection of macrophages was essentially as described (Hasan et al. Positive clones were expanded. Mycobacteria were labeled in 7H9 medium supplemented with 10% OADC Middlebrook containing 5 Ci/ml 35S-methionine/cysteine mix (Promix). and the pellets were discarded.
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