• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Apr 29, 2003; 100(9): 5437–5442.
Published online Apr 17, 2003. doi:  10.1073/pnas.0737613100
PMCID: PMC154363
Microbiology

Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest

Abstract

The tubercle bacillus parasitizes macrophages by inhibiting phagosome maturation into the phagolysosome. This phenomenon underlies the tuberculosis pandemic involving 2 billion people. We report here how Mycobacterium tuberculosis causes phagosome maturation arrest. A glycosylated M. tuberculosis phosphatidylinositol [mannose-capped lipoarabinomannan (ManLAM)] interfered with the phagosomal acquisition of the lysosomal cargo and syntaxin 6 from the trans-Golgi network. ManLAM specifically inhibited the pathway dependent on phosphatidylinositol 3-kinase activity and phosphatidylinositol 3-phosphate-binding effectors. These findings identify ManLAM as the M. tuberculosis product responsible for the inhibition of phagosomal maturation.

The molecular mechanisms controlling phagolysosome biogenesis are of significance both as fundamental membrane trafficking processes and as targets affected by microbes parasitizing host phagocytic cells (1). These two aspects are interrelated, because bacterial pathogens that affect vesicular trafficking provide a means of dissecting the phagosomal maturation pathway. One of the model systems in this context is the mycobacterial phagosome (2). Mycobacterium tuberculosis is a highly adapted pathogen that infects and parasitizes macrophages. The ability of this bacterium, also referred to as the tubercle bacillus, to multiply and persist in the host phagocytic cells is central to its unrivaled notoriety as a pathogen infecting >2 billion people worldwide and causing close to 3 million deaths annually (3). No toxins with a discrete, defined function have been identified in this pathogen thus far despite over a century of research. Nevertheless, certain properties of M. tuberculosis, such as the ability to survive and persist in macrophages, have been singled out as the marquee determinants of its pathogenicity (2, 4).

The persistence of the tubercle bacillus in macrophages relies on an arrest of mycobacterial phagosome maturation (2, 57), a phenomenon referred to in the classical literature as the inhibition of phagosome-lysosome fusion (4). The phagosome maturation arrest allows M. tuberculosis to avoid degradative and antigen-presenting organelles in the host macrophage. The major defining features of the unique M. tuberculosis phagosomal compartments (MPC) are fewer or less-active H+-ATPase molecules (6) and a conspicuous absence of the mature lysosomal hydrolases such as cathepsin D (5, 8). Mycobacterial phagosomes also show aberrant trafficking of plasma membrane markers (5), and “taco” (9), a factor identical to the previously characterized generic phagocytosis protein coronin (10) that seems to be important for the uptake of mycobacteria (11). The abnormal clearance of these early phagosomal markers represents a consequence rather than the cause of the maturation arrest (11).

Organelle biogenesis and maintenance of compartmental integrity in eukaryotic cells depends on multicomponent membrane trafficking systems including vesicle tethering and fusion machinery. The basal apparatus needed for membrane docking and fusion is composed of a complex of cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) molecules contributed by both the donor and acceptor membranes (12, 13). In combination with other regulatory factors, e.g., small GTPases from the Rab family (12) and their effectors such as the membrane tethering molecule early endosome autoantigen 1 (EEA1) (14), SNAREs define the preferred organellar fusion events in vivo (13). We reasoned that the acquisition, retention, or exclusion of compartment-specific SNAREs determines the permissive phagosomal interactions with other organelles in the process of phagosome biogenesis and maturation. Here we report a critical SNARE alteration associated with M. tuberculosis phagosome maturation arrest that contributes to the isolation of mycobacterial phagosomes from the sorting organelles that deliver membrane and luminal components necessary for conversion of a phagosome into the phagolysosome. We also report the identification of a glycosylated phosphatidylinositol product of virulent M. tuberculosis as a cis-acting inhibitor interfering with a sorting pathway that delivers lysosomal hydrolases and H+-ATPase from the trans-Golgi network (TGN) to the phagosome.

Materials and Methods

Cell and Bacterial Culture, Treatment with Inhibitors, and Phagosome Purification.

J774 cells were maintained in DMEM/5% FBS. M. tuberculosis var. bovis bacillus Calmette–Guérin harboring phsp60-gfp or phsp60-dsRed was grown in Middlebrook 7H9 broth. J774 cells were treated with brefeldin A (BFA, 5 μg/ml) for 1 h at 37°C or with 100 nM wortmannin 10 min after phagocytosis (15). Phagocytosis was synchronized for 1 h at 4°C followed by a shift to 37°C. Phagosomes were isolated and characterized as described (7).

Preparation of Lipoarabinomannan-Coated Latex Beads.

Latex beads were coated for 2 h at 37°C in 50 mM NaHCO3, pH 9.6, with purified mannose-capped lipoarabinomannan (ManLAM) or phosphatidylinositol mannoside (PIM) from virulent M. tuberculosis as described (15, 16). The glycolipids were prepared by J. Belisle (Colorado State University, Fort Collins).

Microinjection Studies.

Microinjections of blocking antibody against EEA1 (4 mg/ml) with FITC-dextran Mr 10,000 (1 mg/ml) (15) were performed at 0.58 psi (1 psi = 6.89 kPa) for 0.2 sec by using an Eppendorf Injectman system followed by recovery for 2 h.

Fluorescence Microscopy.

Macrophages were fixed (3.7% paraformaldehyde), permeabilized (0.2% saponin), and incubated with primary antibody followed by secondary Alexa 568- or Alexa 488-conjugated antibody. LysoTracker red DND-99 staining was carried out as described (15). Colocalization was determined by unbiased counting (15, 17) meeting published morphological characteristics (15). Colocalization was quantitated by evaluating ≥100 phagosomes from four separate monolayers per experimental point.

7-Nitrobenzofurazan (NBD)-C6-Sphingomyelin Acquisition.

NBD-C6-sphingomeylin acquisition was monitored as described (18). NBD-C6-ceramide (Molecular Probes) was complexed with 0.034% defatted BSA in DMEM (DMEM-Ceramide) to generate an equimolar ratio of ceramide and BSA (5 μM each) (19). Macrophages were infected with latex beads or M. bovis bacillus Calmette–Guérin-dsRed. After 10 min chase, cells were incubated with DMEM-Ceramide for 30 min at 4°C. Before shifting to 37°C, the medium was replaced with DMEM containing excess BSA (0.34%) to back-exchange probe transported to the plasma membrane.

Western Blot Analysis.

Equal amounts of MPC and latex bead phagosomal compartments (LBC) (by protein) were separated by SDS/PAGE, transferred to poly(vinylidene difluoride) membranes, and probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and visualization was performed by using the ECL Western blotting system.

Antibodies.

The antibodies used were immature cathepsin D (Transduction Laboratories, Lexington, KY), furin (Santa Cruz Biotechnology), lysobisphosphatidic acid (from J. Gruenberg, University of Geneva, Geneva), lysosome-associated membrane protein (LAMP, Development Studies Hybridoma Bank, Iowa City), Rab 9 (from S. Pfeffer, Stanford University, Stanford, CA), Rab 11 (Transduction Laboratories), syntaxin 3 (from A. Hubbard, Johns Hopkins University, Baltimore), syntaxin 6 (StressGen Biotechnologies, Victoria, Canada, and Transduction Laboratories), syntaxin 8 (from W. Hong, Institute of Molecular and Cell Biology, Singapore), syntaxin 13 (from R. Scheller and R. Prekeris, Stanford University), and V0-16kDa (16-kDa subunit of the vacuolar H+-ATPase, from M. Skinner, University of Guelph, Guelph, ON, Canada).

Statistical Analysis.

Statistical significance was calculated by using Fisher's protected least significant difference post hoc test (ANOVA) (SUPERANOVA 1.11, Abacus Concepts, Berkeley, CA).

Results

SNARE Dynamics on Phagosomes.

Isolated LBC and purified mycobacterial phagosomes (MPC), prepared from infected macrophages as described (7, 15), were probed for plasma membrane and endosomal SNAREs. Both LBC and MPC contained but gradually cleared syntaxin 3, a plasma membrane SNARE (Fig. (Fig.11A). Syntaxin 13, associated with endosomal recycling (20), was present on phagosomes and was cleared slowly from LBC and somewhat faster from MPC (Fig. (Fig.11 A and B Inset). Syntaxin 8, a target SNARE that overlaps with Rab 5 on early endosomes (21) and plays a functional role in fusion at a later endosomal stage (22), was examined also. Syntaxin 8 was acquired by both MPC and LBC. Relative to LBC, MPC displayed a delay in the early acquisition of syntaxin 8 (Fig. (Fig.11A). These observations show that phagosomes undergo remodeling involving plasma membrane and endosomal SNAREs and that some of these processes are preserved on mycobacterial phagosomes, albeit with exceptions (e.g., the early time points with syntaxin 8).

Figure 1
Exclusion of the trafficking regulators syntaxin (Syn) 6 and Rab 9 from mycobacterial phagosomes: interrupted communication with the TGN. (A) Western blot analysis of isolated LBC and purified MPC. LBC and MPC (5 μg of protein) were analyzed with ...

Exclusion of Syntaxin 6 from Mycobacterial Phagosomes.

Syntaxin 6 participates in the vesicular traffic between the TGN and the endocytic pathway (23, 24). The bulk of syntaxin 6 in macrophages colocalized with the TGN marker furin (Fig. (Fig.11 CE). Latex bead phagosomes gradually accumulated syntaxin 6 over time (Fig. (Fig.11 A and B, LBC). In contrast, syntaxin 6 was completely absent from mycobacterial phagosomes at all time points (Fig. (Fig.11 A and B, MPC). MPC did not acquire syntaxin 6 even 24 h postformation (Fig. (Fig.11B). Syntaxin 6 acquisition was also examined by immunofluorescence microscopy (Fig. (Fig.11 FJ). Latex bead phagosomes accumulated syntaxin 6, forming distinct ring structures (Fig. (Fig.11H). In contrast, syntaxin 6 was excluded from MPC (Fig. (Fig.11 I and J).

The exclusion of syntaxin 6 from the mycobacterial phagosome suggests that, in contrast to the model phagosomes (LBC), MPC do not fully communicate with TGN, a major biosynthetic and sorting organelle in the cell. In support of this interpretation was the absence on MPC of Rab 9 (Fig. (Fig.11K), a small GTPase controlling mannose 6-phosphate receptor (M6PR) recycling from the endocytic organelles back to the TGN (25). In contrast to Rab 9, both MPC and LBC acquired Rab 11 (Fig. (Fig.11K), a small GTPase controlling recycling from endocytic organelles to the plasma membrane (26). The late appearance of Rab 9 on LBC is consistent with a slow maturation of hydrophobic latex beads into phagolysosomes, which is normally completed only after several hours (27). The reported exclusion of cathepsin D (28), M6PR (29), and H+-ATPase (6) from mycobacterial phagosomes now can be explained by a block in trafficking between the TGN and MPC, because these markers originate in the TGN.

Inhibition of Phagosomal Interactions with the Biosynthetic Pathway Alters Phagosome Maturation.

BFA induces disruption of the Golgi and results in the inhibition of cargo traffic to the endosomal network (30). Acquisition of syntaxin 6 by latex bead phagosomes was reduced in BFA-treated macrophages (Fig. (Fig.11L), which indicates that syntaxin 6 acquisition by phagosomes depends on a functional Golgi apparatus. BFA also reduced (see Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org) the amount of the 43-kDa immature form of cathepsin D in phagosomes by 70% (Fig. 5, which is published as supporting information on the PNAS web site), indicating disruption of the delivery of the lysosomal hydrolases from the TGN to the phagosome. Interference with the biosynthetic pathway also precluded delivery of the 16-kDa transmembrane subunit of the V0-ATPase (V0-p16) (Fig. 6, which is published as supporting information on the PNAS web site) and accumulation of the late endosome-specific marker lysobisphosphatidic acid (Fig. 5). As a control, the recruitment of the endosomal SNARE syntaxin 8 was not affected in cells treated with BFA (Fig. (Fig.11L), showing that the BFA-mediated effect was specific for the trafficking of the TGN SNARE syntaxin 6 and the cargo molecule cathepsin D to the phagosome.

In contrast to the loss of syntaxin 6, LBC from BFA-treated cells acquired LAMP-2 and syntaxin 8 identically to LBC in untreated cells (Fig. (Fig.11L). These observations indicate that BFA treatment specifically interrupts the interactions between the TGN and newly formed phagosomes, whereas LAMP-2 delivery (from endosomes) remains unaltered. This is consistent with a finding that BFA treatment does not affect the acquisition of the early endosomal markers such as syntaxin 13 (31). These observations affirm the presence of two independent pathways contributing to phagosomal interactions, one deriving from the endosome and the other coming (directly or via an intermediary compartment) from the TGN. The contribution of the BFA-sensitive pathway to proteolytic function of phagosomal organelles was also investigated by monitoring fluorescence of biotinylated FITC-casein bound to avidin beads after phagocytosis in BFA-treated and control macrophages (Fig. 7, which is published as supporting information on the PNAS web site). A quantitative analysis of casein degradation and removal from phagosomes, as monitored by reduction in FITC fluorescence associated with phagosomes, was reduced by 50% (P < 0.05) in BFA-treated cells demonstrating the functional significance of traffic from the biosynthetic pathway to phagosomes.

Phagosomal Acquisition of Golgi-Derived Sphingomyelin.

Hackstadt et al. (18) reported the acquisition of Golgi-derived sphingomyelin by Chlamydia trachomatis inclusion bodies. Following established protocols (18), macrophages were loaded with the membrane-permeant precursor C6-ceramide conjugated to the fluorophore NBD, which is converted into NBD-sphingomyelin in the Golgi apparatus (19). The delivery of NBD-C6-sphingomyelin to latex bead phagosomes followed a linear course (1–4 h) (Fig. (Fig.22 A, D, G, and J). The fluorescence label in this experiment was delivered from the Golgi and not the plasma membrane (e.g., via endosomes), because the cells were incubated in the presence of defatted BSA, which extracts sphingomyelin from the plasma membrane (18, 32). The delivery of NBD-C6-sphingomyelin to phagosomes was reduced significantly (35% vs. 70%) by treating the cells with BFA (Fig. (Fig.22 B, E, H, and K). Mycobacterial phagosomes displayed similar properties to that of latex bead phagosomes in BFA-treated cells (Fig. (Fig.22 C, F, I, and K). These observations are consistent with a maturation block preventing the delivery of material from the TGN to the M. tuberculosis phagosome.

Figure 2
Evidence for a sorting pathway between TGN and phagosomes and its disruption by mycobacteria. Shown are live epifluorescent microscopy images of phagosomal acquisition of NBD-C6-sphingomyelin generated from NBD-C6-ceramide in the Golgi apparatus. (A ...

M. tuberculosis ManLAM Inhibits the Accumulation of Syntaxin 6 and Cathepsin D by Latex Bead Phagosomes.

M. tuberculosis produces an array of bioactive lipids that have been implicated, with varying capacities, in pathogenesis of tuberculosis (33). The most prevalent class of compounds, in addition to mycolic acids that dominate the mycobacterial cell wall, are the modified phosphoinositides closely mimicking mammalian phosphatidyl-myo-inositols (Fig. (Fig.33 AC; ref. 34). M. tuberculosis glycosylated phosphoinositides, including the heavily glycosylated ManLAM (Fig. (Fig.33A) and its mono- and dimannosylated precursor PIM (Fig. (Fig.33B), have been reported to intercalate into endomembranes of the infected macrophage (35). It was shown recently that ManLAM inhibits acquisition of EEA1 (15). We tested whether ManLAM interfered with the TGN-to-phagosome sorting pathway uncovered here by examining the acquisition of syntaxin 6 and immature cathepsin D (Fig. (Fig.3).3). ManLAM, purified from virulent M. tuberculosis H37Rv, and its biosynthetic precursor PIM were used to coat latex beads according to established procedures (16). Phagosomes containing ManLAM beads recruited less syntaxin 6 relative to control beads (Fig. (Fig.33 D, E, H, I, and L). Unlike ManLAM, coating latex beads with PIM did not reduce syntaxin 6 acquisition (Fig. (Fig.33M). This difference was not due to a less-efficient coating with PIM, because latex beads coated with PIM exhibited biological activity manifested by increased acquisition of syntaxin 4 (Fig. (Fig.33M). This observation indicates that ManLAM but not its less glycosylated precursor PIM specifically interferes with syntaxin 6 acquisition.

Figure 3
M. tuberculosis glycosylated phosphatidylinositol ManLAM inhibits delivery of syntaxin 6 and immature lysosomal hydrolase delivery to phagosomes. (A) Chemical structure of M. tuberculosis glycosylated phosphatidylinositol ManLAM. A, arabinose; M, mannose. ...

ManLAM-coated beads also accumulated less immature cathepsin D relative to uncoated beads (Fig. (Fig.33 F, G, and JL), demonstrating that ManLAM directly interferes with the delivery of the lysosome-destined cargo such as M6PR-bound lysosomal hydrolases. Similar to the effects of ManLAM on syntaxin 6 acquisition, this glycosylated phosphatidylinositol inhibited immature cathepsin D delivery by 40% (Fig. (Fig.33L). The effect of ManLAM on syntaxin 6 recruitment and immature cathepsin D accumulation cannot be attributed to alterations in phagocytosis of beads, because ManLAM-coated beads recruited syntaxin 8 at levels indistinguishable from uncoated control beads (data not shown). These findings are consistent with a model in which M. tuberculosis glycosylated phosphatidylinositol ManLAM acts as an inhibitor, causing a block in the syntaxin 6-dependent delivery of cargo destined for phagolysosomal compartments.

ManLAM Inhibits the Sorting Pathway from TGN to Phagosomes Dependent on Phosphatidylinositol 3-Kinase (PI3-Kinase) and EEA1.

In considering potential mechanisms of the interference of mycobacterial phosphatidylinositides with phagosomal maturation, we investigated the role of PI3-kinase. PI3-kinase is critical for the delivery of cathepsin D to endosomal organelles (36). Cells were allowed to phagocytize latex beads for 10 min, treated with the PI3-kinase inhibitor wortmannin, and tested for the delivery of syntaxin 6 to phagosomes. Syntaxin 6 acquisition by LBC was inhibited by wortmannin (Fig. (Fig.44 A and B). The effect of wortmannin was limited to syntaxin 6, because levels of syntaxin 8, syntaxin 13, and LAMP-2 were not reduced (Fig. (Fig.44 A and B). Wortmannin did not reduce LAMP accumulation by LBC (Fig. (Fig.44A), in keeping with phagosomes intersecting with at least two distinct pathways: (i) in the first pathway, syntaxin 6 controls post-Golgi delivery of M6PR-bound hydrolytic enzymes in a PI3-kinase-dependent manner (36, 37); and (ii) in the second pathway, phagosomes receive marker, such as LAMPs, in a PI3-kinase-independent manner (37), possibly indirectly via endosomes. Collectively, our data demonstrate that mycobacteria interfere with the syntaxin 6-dependent trafficking from the TGN, primarily affecting the acquisition of membrane and cargo components, the delivery of which depends on PI3-kinase activity.

Figure 4
ManLAM interferes with the wortmannin-sensitive trafficking pathway from TGN to phagosomes. (A) Western blot analysis of purified LBC. Cultures were treated with 100 nM wortmannin (WM) 10 min after bead uptake by J774 cells and incubated with the inhibitor ...

Syntaxin 6 interacts with EEA1 (38), a tethering molecule, which in turn is recruited to organelles via association of its FYVE domain with the phosphatidylinositol-3-phosphate (PI3P), generated after activation of PI3-kinase on membranes that are ear-marked for fusion (39). We reasoned that if the EEA1-syntaxin 6 interaction is an essential prelude to membrane tethering and fusion involving phagosomes, then microinjection of an inhibitory EEA1 antibody would interfere with the formation or function of the syntaxin 6-EEA1 complexes and affect acquisition of syntaxin 6 by phagosomes. Latex bead phagosomes displayed a marked reduction in syntaxin 6 accumulation in cells injected with anti-EEA1 relative to phagosomes in control cells (Fig. (Fig.44C). Microinjecting cells with control rabbit IgG did not reduce syntaxin 6 accumulation relative to uninjected cells (Fig. (Fig.44C). Anti-EEA1 antibody also reduced immature cathepsin D staining (Fig. (Fig.44C). Collectively, these studies demonstrate that PI3-kinase, syntaxin 6, and EEA1 (Fig. (Fig.44D) play a role in the delivery of lysosomal components to the phagosomes. The M. tuberculosis product ManLAM interferes with this pathway and blocks delivery of cargo from TGN to the mycobacterial phagosome.

Discussion

Organellar membrane lipid domains have been shown to play a role in M. tuberculosis phagosome biogenesis (15, 40). Recently, a specific host cell phosphatidylinositol, PI3P, has been implicated in phagosomal maturation in macrophages (15, 41, 42). Because M. tuberculosis produces modified phosphatidylinositols that closely resemble the mammalian lipid equivalents (Fig. (Fig.33 AC), we tested their effects on phagosome maturation. Here we identified the glycosylated M. tuberculosis phosphatidylinositol ManLAM as an inhibitor made by this organism responsible for a block in delivery of lysosomal constituents from the TGN to mycobacterial phagosomes. These findings represent identification of a specific M. tuberculosis product as an inhibitor of a discrete intracellular trafficking pathway.

The arrest of M. tuberculosis phagosome maturation is one of the best defined pathogenic determinants in tuberculosis. This phenomenon has been modeled previously based on presumed abnormal interactions with the endocytic pathway, characterized by the exclusion of late endosomal and lysosomal markers such as H+-ATPase (6), M6PR (29), and cathepsin D (5, 43). However, these essential effector components of the late endosomal and lysosomal organelles, as shown here, are delivered to the phagosomes from the TGN. Our data demonstrate that the exclusion of syntaxin 6 but not other endosomal syntaxins reflects the presence of a block in trafficking between the TGN and the M. tuberculosis phagosome that is not indirect, e.g., via endosomes. This is an important distinction, because the trafficking between the TGN and early endosomes is also regulated by syntaxin 6 (24). Because syntaxin 6 is present on model phagosomes and absent from mycobacterial phagosomes, these findings define the TGN-to-phagosome trafficking pathway as the sorting mechanism targeted by ManLAM.

The exclusion of syntaxin 6 from MPC precludes efficient delivery of lysosomal enzymes and proton-pump subunits to mycobacterial phagosomes. Syntaxin 6 exclusion from MPC also provides the mechanism for the absence of M6PR from mycobacterial phagosomes (29) and prevention of the assembly of a complete and functional H+-ATPase complex (6). Because these are salient characteristics of the M. tuberculosis phagosome that render it compatible with the long-term survival of this pathogen in the macrophage, the interference of ManLAM with the sorting pathway between the TGN and the phagosome is a critical event in M. tuberculosis phagosome maturation arrest.

Our studies show direct interactions of phagosomes with the biosynthetic pathway in delivery of cargo from the TGN. This may seem to challenge the phagosome–endosome interaction model as the proposed pathway of macrophage maturation (44). However, the two models are not necessarily in conflict, because our observations indicate that there are at least two distinct pathways for delivery of lysosomal proteins and membrane components to the phagosomes. (i) The first one delivers M6PR-bound hydrolytic enzymes, in a manner that is sensitive to wortmannin and depends on the PI3-kinase hVPS34 (36). (ii) The second pathway is PI3-kinase-independent and is responsible for the delivery of the integral membrane proteins such as LAMPs (37). This dichotomy is illustrated further by the findings that phagosomal lysobisphosphatidic acid acquisition and phagosome acidification are reduced markedly by wortmannin (15), whereas LAMP levels are not affected. Our data are consistent with the inhibition of the PI3-kinase-dependent pathway from the TGN as having a major role in the M. tuberculosis phagosome maturation arrest. This does not preclude additional phagosomal interactions with the endocytic pathway, which may be operational or potentially affected by mycobacteria, as evidenced by a delay in MPC acquisition of syntaxin 8 (see Fig. Fig.11A). At present, it is not known whether the observed anomalous acquisition of syntaxin 8 during early time points has any effects on MPC maturation. However, because it has been shown that syntaxin 8 plays a functional role in late endosomal fusion (22), its delayed acquisition may contribute to the mycobacterial phagosome maturation block.

In this study we found that phagosomes containing latex beads coated with the M. tuberculosis cis-acting inhibitor ManLAM undergo reduced syntaxin 6 acquisition. These results, and additional studies showing the importance of PI3-kinase and EEA1 (15), indicate that ManLAM alters phagosome maturation by blocking the syntaxin 6-dependent delivery of cargo from the TGN. This pathway depends on (i) PI3P generation and accessibility (15), (ii) the tethering factor EEA1, which recognizes and binds to PI3P on organellar membranes via its FYVE domain (45), and (iii) EEA1–syntaxin 6 interactions (38). The M. tuberculosis glycolipid ManLAM interferes with this cascade (Fig. (Fig.44D). Based on this information, ManLAM biosynthesis can be considered as a prime target for development of new antituberculosis drugs, with intracellular localization-altering properties, that will empower the innate and immune defenses of the host to eliminate intracellular M. tuberculosis.

Supplementary Material

Supporting Information:

Acknowledgments

We thank J. Belisle, S. Corvera, J. Gruenberg, W. Hong, A. Hubbard, S. Pfeffer, R. Prekeris, R. Scheller, M. Skinner, and P. Tuma, for reagents. The glycolipids were prepared by J. Belisle through support by National Institutes of Health Grant N01-AI-75320. This work was supported by National Institutes of Health Grant AI45148.

Abbreviations

MPC
Mycobacterium tuberculosis phagosomal compartments
SNARE
soluble N-ethylmaleimide-sensitive factor attachment protein receptor
EEA1
early endosome autoantigen 1
TGN
trans-Golgi network
BFA
brefeldin A
ManLAM
mannose-capped lipoarabinomannan
PIM
phosphatidylinositol mannoside
NBD
7-nitrobenzofurazan
LAMP
lysosome-associated membrane protein
M6PR
mannose 6-phosphate receptor
PI3-kinase
phosphatidylinositol 3-kinase
PI3P
phosphatidylinositol-3-phosphate

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

References

1. Meresse S, Steele-Mortimer O, Moreno E, Desjardins M, Finlay B, Gorvel J P. Nat Cell Biol. 1999;1:E183–E188. [PubMed]
2. Russell D G, Mwandumba H C, Rhoades E E. J Cell Biol. 2002;158:421–426. [PMC free article] [PubMed]
3. Dye C, Scheele S, Dolin P, Pathania V, Raviglione M C. J Am Med Assoc. 1999;282:677–686. [PubMed]
4. Armstrong J A, Hart P D. J Exp Med. 1971;134:713–740. [PMC free article] [PubMed]
5. Clemens D L, Horwitz M A. J Exp Med. 1995;181:257–270. [PMC free article] [PubMed]
6. Sturgill-Koszycki S, Schlesinger P H, Chakraborty P, Haddix P L, Collins H L, Fok A K, Allen R D, Gluck S L, Heuser J, Russell D G. Science. 1994;263:678–681. [PubMed]
7. Via L E, Deretic D, Ulmer R J, Hibler N S, Huber L A, Deretic V. J Biol Chem. 1997;272:13326–13331. [PubMed]
8. Sturgill-Koszycki S, Schaible U E, Russell D G. EMBO J. 1996;15:6960–6968. [PMC free article] [PubMed]
9. Ferrari G, Langen H, Naito M, Pieters J. Cell. 1999;97:435–447. [PubMed]
10. Maniak M, Rauchenberger R, Albrecht R, Murphy J, Gerisch G. Cell. 1995;83:915–924. [PubMed]
11. Schuller S, Neefjes J, Ottenhoff T, Thole J, Young D. Cell Microbiol. 2001;3:785–793. [PubMed]
12. Bock J B, Matern H T, Peden A A, Scheller R H. Nature. 2001;409:839–841. [PubMed]
13. Parlati F, Varlamov O, Paz K, McNew J A, Hurtado D, Sollner T H, Rothman J E. Proc Natl Acad Sci USA. 2002;99:5424–5429. [PMC free article] [PubMed]
14. McBride H M, Rybin V, Murphy C, Giner A, Teasdale R, Zerial M. Cell. 1999;98:377–386. [PubMed]
15. Fratti R A, Backer J M, Gruenberg J, Corvera S, Deretic V. J Cell Biol. 2001;154:631–644. [PMC free article] [PubMed]
16. Kang B K, Schlesinger L S. Infect Immun. 1998;66:2769–2777. [PMC free article] [PubMed]
17. Botelho R J, Hackam D J, Schreiber A D, Grinstein S. J Biol Chem. 2000;275:15717–15727. [PubMed]
18. Hackstadt T, Scidmore M A, Rockey D D. Proc Natl Acad Sci USA. 1995;92:4877–4881. [PMC free article] [PubMed]
19. Pagano R E, Martin O C. Biochemistry. 1988;27:4439–4445. [PubMed]
20. Prekeris R, Klumperman J, Chen Y A, Scheller R H. J Cell Biol. 1998;143:957–971. [PMC free article] [PubMed]
21. Subramaniam V N, Loh E, Horstmann H, Habermann A, Xu Y, Coe J, Griffiths G, Hong W. J Cell Sci. 2000;113:997–1008. [PubMed]
22. Antonin W, Holroyd C, Fasshauer D, Pabst S, Von Mollard G F, Jahn R. EMBO J. 2000;19:6453–6464. [PMC free article] [PubMed]
23. Bock J B, Klumperman J, Davanger S, Scheller R H. Mol Biol Cell. 1997;8:1261–1271. [PMC free article] [PubMed]
24. Mallard F, Tang B L, Galli T, Tenza D, Saint-Pol A, Yue X, Antony C, Hong W, Goud B, Johannes L. J Cell Biol. 2002;156:653–664. [PMC free article] [PubMed]
25. Barbero P, Bittova L, Pfeffer S R. J Cell Biol. 2002;156:511–518. [PMC free article] [PubMed]
26. Ullrich O, Reinsch S, Urbe S, Zerial M, Parton R G. J Cell Biol. 1996;135:913–924. [PMC free article] [PubMed]
27. de Chastellier C, Lang T, Thilo L. Eur J Cell Biol. 1995;68:167–182. [PubMed]
28. Clemens D L, Horwitz M A. J Exp Med. 1996;184:1349–1355. [PMC free article] [PubMed]
29. Xu S, Cooper A, Sturgill-Koszycki S, van Heyningen T, Chatterjee D, Orme I, Allen P, Russell D G. J Immunol. 1994;153:2568–2578. [PubMed]
30. Fernandez C J, Haugwitz M, Eaton B, Moore H P. Mol Biol Cell. 1997;8:2171–2185. [PMC free article] [PubMed]
31. Fratti R A, Chua J, Deretic V. J Biol Chem. 2002;277:17320–17326. [PubMed]
32. Scidmore M A, Fischer E R, Hackstadt T. J Cell Biol. 1996;134:363–374. [PMC free article] [PubMed]
33. Brennan P J, Nikaido H. Annu Rev Biochem. 1995;64:29–63. [PubMed]
34. Besra G S, Chatterjee D. In: Tuberculosis: Pathogenesis, Protection, and Control. Bloom B R, editor. Washington, DC: Am. Soc. Microbiol.; 1994. pp. 285–306.
35. Beatty W L, Rhoades E R, Ullrich H-J, Chatterjee D, Heuser J E, Russell D G. Traffic. 2000;1:235–247. [PubMed]
36. Row P E, Reaves B J, Domin J, Luzio J P, Davidson H W. Biochem J. 2001;353:655–661. [PMC free article] [PubMed]
37. Karlsson K, Carlsson S R. J Biol Chem. 1998;273:18966–18973. [PubMed]
38. Simonsen A, Gaullier J M, D'Arrigo A, Stenmark H. J Biol Chem. 1999;274:28857–28860. [PubMed]
39. Simonsen A, Wurmser A E, Emr S D, Stenmark H. Curr Opin Cell Biol. 2001;13:485–492. [PubMed]
40. Gatfield J, Pieters J. Science. 2000;288:1647–1650. [PubMed]
41. Vieira O V, Botelho R J, Rameh L, Brachmann S M, Matsuo T, Davidson H W, Schreiber A, Backer J M, Cantley L C, Grinstein S. J Cell Biol. 2001;155:19–25. [PMC free article] [PubMed]
42. Gillooly D J, Simonsen A, Stenmark H. J Cell Biol. 2001;155:15–17. [PMC free article] [PubMed]
43. Malik Z A, Iyer S S, Kusner D J. J Immunol. 2001;166:3392–3401. [PubMed]
44. Desjardins M. Trends Cell Biol. 1995;1995:183–186. [PubMed]
45. Lawe D C, Patki V, Heller-Harrison R, Lambright D, Corvera S. J Biol Chem. 2000;275:3699–3705. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...