Mechanism of Phagolysosome Biogenesis Block by Viable Mycobacterium Tuberculosis

Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis

Isabelle Vergne*†, Jennifer Chua*†, Hwang-Ho Lee*, Megan Lucas‡, John Belisle‡, and Vojo Deretic*§¶

Departments of *Molecular Genetics and Microbiology and §Cell Biology and Physiology, University of New Mexico School of Medicine, Albuquerque, NM 87131; and ‡Department of Microbiology, Colorado State University, Fort Collins, CO 80523

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved January 26, 2005 (received for review December 23, 2004) Live Mycobacterium tuberculosis persists in macrophage phago- M. tuberculosis growth as a defense mechanism downstream of somes by interfering with phagolysosome biogenesis. Here, using macrophage activation by IFN-␥ (23). four-dimensional microscopy and in vitro assays, we report the In addition to hVPS34 interactions with Rab5, the recruitment of ϩ principal difference between phagosomes containing live and hVPS34 to endomembranes is controlled in macrophages by Ca2 , ϩ dead mycobacteria. Phosphatidylinositol 3-phosphate (PI3P), a calmodulin (CaM), and Ca2 ͞CaM kinase II (24). It has been membrane trafficking regulatory lipid essential for phagosomal shown that M. tuberculosis lipoarabinomannan inhibits cytosolic ϩ acquisition of lysosomal constituents, is retained on phagosomes Ca2 rise during phagocytosis (24, 25). A model of how M. harboring dead mycobacteria but is continuously eliminated from tuberculosis blocks phagosome maturation has emerged, based on phagosomes with live bacilli. We show that the exclusion of PI3P altered hVPS34 recruitment to mycobacterial phagosomes and from live mycobacterial phagosomes can be only transiently re- altered PI3P patterns relative to the model, latex bead phagosomes versed by Ca2؉ fluxes, and that live M. tuberculosis secretes a lipid (12, 14, 24). PI3P is essential for phagosome maturation into the phosphatase, SapM, that hydrolyzes PI3P, inhibits phagosome–late phagolysosome, and inhibition of PI3P production arrests phago- endosome fusion in vitro, and contributes to inhibition of phagosome maturation (11, 16). However, the status of PI3P on phagosomal maturation. somes containing live vs. dead M. tuberculosis is not known. In this work, we show, using live cell imaging, four-dimensional (4D) macrophage ͉ phagosome ͉ tuberculosis ͉ lysosome ͉ phosphatidylinositol confocal microscopy, and in vitro assays, that the principal differ- 3-phosphate ence between phagosomes harboring live or dead mycobacteria is the persistence of PI3P on phagosomes with killed microorganisms vs. the removal of PI3P from phagosomes harboring live bacilli. We he infectious cycle of Mycobacterium tuberculosis rests upon show that, in addition to the known effects of the tubercle bacillus Tthe ability of this potent pathogen to parasitize host mono- on suppressing Ca2ϩ fluxes (26), which in turn affect the recruit- nuclear phagocytic cells (1). In infected macrophages, M. tuber- ment of the PI3K responsible for generating PI3P on endomem- culosis resides within a phagosome that avoids the default branes (24), M. tuberculosis encodes a phosphatase that dephos- maturation pathway leading to phagolysosome formation (2). phorylates PI3P and inhibits phagosome–late endosome fusion. The salient characteristics of the mycobacterial phagosome These findings help explain how live M. tuberculosis maintains the ϩ include (i) paucity of vacuolar H ATPase (3), (ii) attendant phagosome maturation block and avoids lysosomal compartments. inefficient luminal acidification (3); and (iii) inadequate levels of mature lysosomal hydrolases (3, 4). These and additional (4–6) Materials and Methods properties of the M. tuberculosis phagosome promote intracel- Cell and Bacterial Cultures, Plasmid Constructs, Transfection, Micros- lular survival and growth of the tubercle bacilli and help avoid copy, and Immunoblotting. RAW 264.7 cells were maintained in their immunological detection (1). DMEM, 4 mM L-glutamine, and 10% FBS. M. tuberculosis var. The arrest of M. tuberculosis phagosome maturation has been bovis bacillus Calmette–Gue´rin(BCG) was grown in 7H9 broth, studied at the membrane-trafficking level (2), with a focus on the and single-cell suspensions were prepared as described (11). For small GTP-binding proteins, including Rab GTPases (7–9). Rabs survival assays, mycobacteria were grown on 7H11 plates. Myco- direct intracellular trafficking by regulating activity and recruitment bacteria either expressed DsRed or were fluorescently labeled with to organellar membranes of Rab-interacting partners and down- 5mg͞ml Texas red-X in PBS for 1 h. Bacteria were opsonized in stream effectors (10). The initial analyses of Rabs on mycobacterial DME supplemented with 10% FBS for 30 min. Mycobacteria were phagosomes have indicated that the M. tuberculosis phagolysosome heat killed by incubation at 90°C for 10 min before labeling. The biogenesis arrest occurs between the stages controlled by the early plasmid constructs and sources were as follows: P40PX-EGFP (M. endosomal GTPase Rab5 and its late endosomal counterpart Rab7 Yaffe, Massachusetts Institute of Technology, Cambridge); (7). A number of follow-up studies have indicated critical contri- 2xFYVE-EGFP (H. Stenmark, Norwegian Radium Hospital, butions of Rab5 effectors in mycobacterial phagosome maturation Oslo); MTM1-EGFP (J. Dixon, University of Michigan, Ann arrest, with a prominent role for the phosphatidylinositol 3-kinase Arbor), and MTMR3-EGFP (M. Clague, University of Liverpool, (PI3K) hVPS34, its product phosphatidylinositol 3-phosphate Liverpool, England). Macrophage transfection, immunofluorescence microscopy and 4D confocal microscopy were carried out as (PI3P), and an array of PI3P-binding proteins (11–14). PI3P affects described (14). Live cell imaging is detailed in Supporting Text and localization and function of proteins containing the PI3P-binding Movies 1–5, which are published as supporting information on the domains (FYVE, PH, and PX) (15). These proteins in turn execute PNAS web site. M. tuberculosis KatG antibody was from C. Barry various steps in membrane trafficking, endosomal protein sorting, and multisubunit enzyme assembly at the membrane, including phagosomal maturation (11, 16), early endosomal homotypic fusion This paper was submitted directly (Track II) to the PNAS office. (17), delivery of internalized plasma membrane receptors to late Abbreviations: PI3K, phosphatidylinositol 3-kinase; PI3P, phosphatidylinositol 3-phosphate; endosomes (18), formation of internal vesicles within late endoso- 4D, four-dimensional; BCG, bacillus Calmette–Gue´rin; MtCFP, Mycobacterium tuberculosis mal multivesicular bodies involved in termination of signaling H37Rv culture filtrate protein; RFU, relative fluorescence unit; MTM, myotubularin. events (19, 20), and phagocyte NADPH oxidase assembly at the †I.V. and J.C. contributed equally to this work.

membrane (21). PI3P is also important for the execution of the ¶To whom correspondence should be addressed. E-mail: [email protected]. CELL BIOLOGY process of autophagy (22), which has recently been shown to restrict © 2005 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409716102 PNAS ͉ March 15, 2005 ͉ vol. 102 ͉ no. 11 ͉ 4033–4038 Downloaded by guest on October 2, 2021 (National Institutes of Health, Bethesda). Affinity purified rabbit polyclonal antibody was raised against a peptide corresponding to the SapM residues 286–299 by using commercial services.

PI3P Phosphatase Activity. Fluorescent phosphoinositide substrates labeled with C6-NBD were from Echelon Research Laboratories (Salt Lake City). Phosphatase activity was monitored according to Taylor and Dixon (27) with fluorescent substrates in 50 mM ammonium acetate (pH 6.0) and 2 mM DTT for 30 min at 30°C. TLC analysis of reaction products was carried out as described (27). The release of phosphate was quantified by malachite green assay (Upstate Biotechnology, Lake Placid, NY) (28) in 50 mM Tris⅐HCl (pH 7.4) and 0.05% Triton X-100 at 37°C.

Phagosome–Late Endosome Fusion Assay. Phagosomes and late endosomes were purified, and fusion assay was carried out as described (5).

Bacterial Culture Supernatants, Bacterial Extracts, and Purified Pro- teins. Culture supernatant was obtained by filtering through a 0.2-␮m filter. Mycobacterial pellets were homogenized by bead beating. When required, J774 were infected with BCG for2hand lysed, and a postnuclear supernatant was prepared according to Via et al. (7). The postnuclear supernatant was subjected to velocity sedimentation (1,000 ϫ g, 45 min, 4°C) through 15% (wt͞wt) sucrose overlaid on a 50% (wt͞wt) sucrose cushion (7). The material at the 15–50% sucrose interface, containing mycobacterial phagosomes, was collected, and extracts were prepared by bead Fig. 1. PI3P persists on phagosomes containing dead but not live M. tuber- beating. M. tuberculosis H37Rv culture filtrate protein (MtCFP) culosis var. bovis BCG. (A) RAW 264.7 macrophages were transfected with and SapM were prepared as described (29, 30), respectively. Pure P40PX-EGFP, allowed to phagocytose either live or dead (heat-inactivated) MptpA and MptpB (GST fusions) were from A. Koul (31). MtCFP Texas red-labeled BCG, and analyzed by 4D confocal microscopy. Shown is was denatured by heating for 30 min at 95°C. quantification of PI3P positivity of phagosomes containing live or dead BCG (n ϭ 45 live, n ϭ 19 dead). (Insets) GFP fluorescence of the PI3P probe Results and Discussion (grayscale) (Left) and merged images of GFP and red mycobacterial fluores- Persistence of PI3P on Phagosomes Containing Dead Mycobacteria. cence (Right). **, P Ͻ 0.01. (B) Temporal quantification of phagosome fluo- Applying 4D confocal microscopy (14), we compared PI3P levels on rescence intensity relative to fluorescence of the cytosol. R␾/c, ratio between phagosomes harboring either live or dead (heat-killed) M. tuber- phagosome fluorescence intensity and cytosol fluorescence intensity. Shown are R␾/c obtained by 4D microscopy and live imaging of three different culosis var. bovis BCG. RAW 264.7 macrophages were transfected phagosomes harboring dead BCG (filled squares) and three different phago- with constructs encoding GFP fusions to protein domains p40phox somes in cells infected with live BCG (open triangles). (C) Quantification of PX (P40PX-EGFP) or 2xFYVE from Hrs (2xFYVE-EGFP) that fluorescence levels over time, expressed in relative fluorescence units (RFU; specifically bind PI3P (21, 32). Macrophages were infected with subtracted for RFU of the cytosol) of a phagosome with dead (filled squares) Texas red-labeled live or dead M. tuberculosis var. bovis BCG. The or live (open triangles) BCG. (D) Confocal immunofluorescence images of fixed fluorescence intensity of the PI3P probe was imaged over time by specimens containing macrophages infected with live or dead BCG and im- rapid collection of z-stacks composed of dual color confocal optical muno-stained for CD63. (E) Quantification of CD63 staining of phagosomes. sections. The images were analyzed, as described (14): (i) for each time point, the optical section corresponding to the maximum phagosomes with dead mycobacteria (PI3P-positive) matured dif- fluorescence intensity of the mycobacteria was identified; and (ii) ͞ this and additional confocal slices above and below were collapsed ferently. When the late endosomal lysosomal marker CD63 (33) into a single x-y projection. was quantified, 32% of the phagosomes harboring live mycobac- Of the phagosomes containing dead mycobacteria monitored for teria colocalized with CD63, whereas 86% of the phagosomes 60 min, 100% (n ϭ 19) remained positive for the PI3P probe at all containing dead mycobacteria colocalized with CD63 (Fig. 1 D times (Fig. 1A and Movie 1). Of the phagosomes in macrophages and E). infected with live mycobacteria, 18% (n ϭ 39) were scored as PI3P-positive (Fig. 1A and Movie 2). Temporal analysis of the Maintenance of PI3P on Phagosomes Containing Dead Mycobacteria mean fluorescence PI3P probe intensity on phagosomes relative to Is PI3-Kinase-Dependent. Conversion of phosphatidylinositol to PI3P is catalyzed by the type III PI3K hVPS34, a kinase that can be the cytosol (RØ/C) is shown in Fig. 1B; relative fluorescence units (RFU) corrected for RFU of the cytosol are shown in Fig. 1C. The reversibly inhibited by LY294002 (34). RAW 264.7 cells transfected newly formed phagosomes displayed early and transient recruit- with the PI3P GFP probe were treated with 100 ␮M LY294002 and ment of the PI3P GFP probe, as reported (14), concomitant with infected with M. tuberculosis var. bovis BCG. LY294002 did not the mycobacterial uptake by the macrophage. Past this initial inhibit mycobacterial uptake. After a chase period, LY294002 was period, dead and live mycobacteria showed dramatic differences, washed out, and recruitment of the PI3P probe to phagosomes was with the phagosomes containing dead mycobacteria being PI3P- monitored by live microscopy. LY294002 inhibited the generation positive at all times, and with those containing live mycobacteria of PI3P on phagosomes, resulting in a disappearance of PI3P (Fig. becoming and remaining fully PI3P-negative. The RØ/C value for 2A). Upon LY294002 washout (Fig. 2B), PI3P was regenerated on dead mycobacterial phagosomes was maintained Ͼ2, whereas live phagosomes with dead mycobacteria (Fig. 2 A–C and Movie 3). The ͞ mycobacteria declined to an RØ/C of 1 (Fig. 1B). In keeping with the LY294002 addition washout cycle did not change the PI3P- previously established role of PI3P in phagosomal maturation (11, negative status of phagosomes with live mycobacteria (Fig. 2 D–F). 16), phagosomes harboring live mycobacteria (PI3P-negative) and Fig. 2G shows the kinetics of PI3P changes on phagosomes. Thus,

4034 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409716102 Vergne et al. Downloaded by guest on October 2, 2021 Fig. 2. De novo generation and maintenance of PI3P on phagosomes harboring dead mycobacteria. RAW 264.7 cells were transfected with P40PX-EGFP, allowed to phagocytose either live or dead BCG (Texas red-labeled), and analyzed by 4D microscopy by using an UltraView microscope. Cells were treated with PI3K inhibitor (100 ␮M LY294002) as indicated (ϩLY). LY294002 washout (removal of the inhibitor) is indicated in the fluorescent image panels (ϪLY) and the graph (arrow). (Insets) Grayscale images of green (Left) and red (Right) fluorescence of areas with objects indicated by arrows. (A–C) Absence of PI3P probe on dead BCG phagosomes in cells treated with LY294002, and recruitment of PI3P probe upon LY294002 washout. (D–F) PI3P probe status (PI3P-negative) remains unchanged on live BCG phagosomes after a cycle of LY294002 treatment and washout. (G) Time course of phagosomal GFP fluorescence (expressed as percent of the maximum RFU) on dead BCG phagosomes with (open squares) or without (filled squares) LY294002 treatment. Arrow, LY294002 washout.

PI3P generation on dead mycobacterial phagosomes is due to a with MTM1-GFP, were allowed to phagocytose live or dead PI3K activity, whereas live mycobacteria have the capacity to mycobacteria, and the cells were analyzed by 4D confocal micros- continually eliminate PI3P from the phagosomal membrane. copy. MTM1-GFP was recruited to both types of phagosomes (Fig. 7, which is published as supporting information on the PNAS web Increasing Intracellular Ca2؉ Only Transiently Restores PI3P Levels on site, and Movies 4 and 5). MTM1-GFP was present only early on Phagosomes with Live Mycobacteria. There is a correlation be- and rapidly disappeared from either type of phagosomes. Similar tween mycobacterial entry into the macrophage and inhibition of phenomena were observed with the PI3P phosphatase MTMR3 cytosolic Ca2ϩ rise (26). The Ca2ϩ binding protein calmodulin (36). Both MTM1 and MTMR3 peaked and then desorbed from (CaM) and its effector Ca2ϩ͞CaM kinase II recruit the PI3K phagosomes at early time points. Neither MTM1 nor MTMR3 was hVPS34 responsible for PI3P production to organellar mem- connected to the later period of intense differences in PI3P levels brane (24). We tested whether increasing intracellular Ca2ϩ can between live and dead mycobacterial phagosomes. Thus, host cell restore PI3P levels on phagosomes with live mycobacteria. RAW PI3P phosphatases, MTM1 and MTMR3, cannot explain the 264.7 cells transfected with the PI3P probe and phagocytosing observed differential removal of PI3P from phagosomes harboring live mycobacteria were treated with the Ca2ϩ ionophore A23187 live mycobacteria. Use of a bacteriostatic antibiotic, chloramphen- as described (26), and analyzed by 4D confocal microscopy. Fig. icol, permitted a demonstration that a bacterial product was 6, which is published as supporting information on the PNAS responsible for controlling the PI3P levels on mycobacterial phago- web site, shows a transient spike in PI3P increase on a phagosomes (Fig. 8, which is published as supporting information on the some containing live mycobacteria in cells treated with A23187. PNAS web site). Treatment of BCG with chloramphenicol in- The PI3P spike occurred only in 10% of all phagosomes ob- creased PI3P positivity of mycobacterial phagosomes (Fig. 8 A, B, served, and the majority of phagosomes did not show PI3P and D). A washout of chloramphenicol (which resulted in resump- increase, despite the fact that Ca2ϩ rise was detected in Ͼ95% tion of mycobacterial growth; Fig. 8E) reversed the PI3P status to of the cells treated with the ionophore (Fig. 6 K–M). Thus, Ca2ϩ the low levels seen with untreated live mycobacteria (Fig. 8 C alone cannot explain the difference in steady-state levels of PI3P and D). on phagosomes harboring dead vs. live mycobacteria. M. tuberculosis Secretes a PI3P 3-Phosphatase. In lieu of explanations Host PI3P Phosphatases and Absence of PI3P on Live Mycobacterial based on Ca2ϩ or host cell phosphatases for differences in PI3P Phagosomes. Experiments with LY294002 addition͞washout (Fig. levels on phagosomes with live vs. dead mycobacteria, we tested 2) indicate that PI3P levels on phagosomes represent a balance whether a mycobacterial enzymatic activity was responsible for the between PI3P synthesis and its turnover. We therefore tested the lack of PI3P on phagosomes harboring live bacilli. Culture filtrate possibility that differential recruitment of a host PI3P-specific protein from virulent M. tuberculosis H37Rv, MtCFP, was incu- 3-phosphatase can explain differences between phagosomes har- bated in an in vitro assay for PI3P phosphatase, and phosphoino-

boring live vs. dead bacilli. Myotubularin 1 (MTM1) specifically sitide products were resolved by TLC. Incubation with MtCFP CELL BIOLOGY dephosphorylates PI3P (35). RAW 264.7 macrophages, transfected resulted in a dose-dependent conversion of PI3P into its dephos-

Vergne et al. PNAS ͉ March 15, 2005 ͉ vol. 102 ͉ no. 11 ͉ 4035 Downloaded by guest on October 2, 2021 Fig. 3. M. tuberculosis secretes PI3P phosphatase SapM. (A) TLC (UV fluorescence) of reaction products when PI3P is incubated in the presence of MtCFP. Di-C6-NBD6-PI3P (1 ␮g) was incubated with different concentrations of MtCFP or boiled MtCFP. Lane 1, MtCFP incubated without Di-C6-NBD6-phosphoinositides; lane 2, Di-C6-NBD6-PI3P incubated without CFP; lane 3, Di-C6-NBD6-PI (product of PI3P hydrolysis) standard; lanes 4–6, Di-C6-NBD6-PI3P incubated with different concentrations of MtCFP; lane 7, Di-C6-NBD6-PI3P incubated with heat-inactivated MtCFP. (B) TLC (UV fluorescence) of reaction products after incubation of PI3P with SapM. Lane 1, Di-C6-NBD6-PI standard; lane 2, Di-C6-NBD6-PI3P incubated without SapM; lanes 3–7, Di-C6-NBD6-PI3P incubated with different concentrations of SapM. (C) PI3P phosphatase activity in MtCFP (40 ␮g͞ml) determined by using malachite green assay. Error bars represent SEM. (D) Comparison of SapM (1.5 ␮g͞ml) and MtCFP (40 ␮g͞ml) phosphatase specificity for phosphatidylinositol monophosphates (PI3P, PI4P, PI5P) determined by using malachite green assay. Error bars represent SEM. (E) Immunoblot comparison of SapM and KatG in MtCFP, bacterial cell extracts (BCG pellet), BCG culture supernatant (BCG Sup), and BCG phagosomes after 2-h infection of J774 macrophages.

phorylation product, phosphatidylinositol (PI) (Fig. 3A). Heat (Fig. 3C). The preferred SapM substrate among phosphatidyl- inactivation of MtCFP abrogated its ability to dephosphorylate inositol monophosphates was PI3P, matching the substrate spec- PI3P (Fig. 3A). The breakdown of PI3P by MtCFP was quantified ificity profile of MtCFP (Fig. 3D). When a panel of phosphatase by using a colorimetric assay (28) (Fig. 3B). These results demon- inhibitors was tested, MtCFP and purified SapM showed iden- strate that M. tuberculosis-secreted proteins contain PI3P phospha- tical sensitivity patterns with PI3P as a substrate (Fig. 9, which tase activity. is published as supporting information on the PNAS web site). We next considered candidate gene products encoded by the Thus, SapM is an M. tuberculosis PI3P phosphatase accounting M. tuberculosis genome showing similarities to the active site in for at least a part of the PI3P phosphatase activity in MtCFP. mammalian myotubularins. The characterized myotubularins Antibodies raised against a SapM peptide showed that SapM was are lipid phosphatases (with MTM1 having very poor tyrosine͞ present in the CFP from M. tuberculosis and that it was enriched dual specificity protein phosphatase activity), showing prefer- in BCG culture supernatant relative to a representative bacterial ence for monophosphorylated substrates such as PI3P, and cytoplasmic protein, KatG, enriched in mycobacterial pellet contain the Cys-X5-Arg motif, including additional Asp residues (Fig. 3E). Furthermore, SapM contains a typical leader peptide within their active site (37). The closest match with this motif was proteolytically removed during its secretion͞maturation (30). found in the M. tuberculosis protein MptpB (encoded by SapM was present in vivo during macrophage infection. Macro- Rv0153c), which was recently described as a secreted tyrosine phages were infected, and phagosomes werre prepared by col- phosphatase (31). When purified GST-MptpB was tested, no lecting the material from the first sucrose gradient following the PI3P phosphatase activity could be detected (data not shown). published phagosome purification procedure (7). Mycobacterial A second secreted tyrosine phosphatase, MptpA (encoded by Rv phagosomes from such preparations were positive for SapM 2234), also showed no PI3P activity, thus ruling out these (Fig. 3E). proteins as possible candidates. Another secreted acid phosphatase activity has been described in M. tuberculosis culture filtrate M. tuberculosis PI3P Phosphatase Is Required for Reduction of PI3P (38) and was later purified and characterized as a nonspecific Levels on Phagosomes and Inhibition of Phagosome Maturation acid phosphatase (30). Although it was concluded in the initial Containing Live Mycobacteria. We used the combined property of report on SapM that it had no activity against phospholipids, molybdate to inhibit SapM (Fig. 9) and its inability to cross host based on the absence of activity with phosphatidylcholine, cell membrane (39, 40) to inhibit specifically secreted bacterial phosphatidylethanolamine, and phosphatidic acid (30), when products during mycobacterial uptake by macrophages. Infec- purified M. tuberculosis SapM was tested with PI3P as a sub- tion of macrophages in the presence of molybdate resulted in strate, it showed strong activity comparable with that of MtCFP increased PI3P positivity of mycobacterial phagosomes (Fig. 4

4036 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409716102 Vergne et al. Downloaded by guest on October 2, 2021 Fig. 4. M. tuberculosis var. bovis BCG-secreted PI3P phosphatase is responsible for removal of PI3P from phagosomes harboring live mycobacteria. RAW 264.7 cells were transfected with P40PX-EGFP, allowed to phagocytose either live or dead mycobacteria (Texas red-labeled BCG), and analyzed by 4D microscopy. Infection with BCG was carried out in the presence or absence of sodium molybdate (1 mM) during phagocytosis and subsequent imaging. (A–C) Lack of PI3P probe recruitment to phagosomes harboring live BCG. (D–F) Recruitment of PI3P probe to phagosomes containing dead BCG. (G–I) Recruitment of PI3P probe on phagosomes containing live BCG treated with sodium molybdate. (J) Fraction (%) of live BCG phagosomes (n ϭ 45) and live BCG phagosomes treated with sodium molybdate (n ϭ 18) that recruited the PI3P probe. *, P ϭ 0.02. (K) Temporal analysis and quantification of phagosome fluorescence intensity relative to cytosol fluorescence (R␾/c). R␾/c were obtained by processing 4D microscopy images of live cells with phagosomes: harboring dead BCG (filled squares), harboring live BCG-treated with sodium molybdate (filled triangles), or with live BCG (open triangle). (L) Colony-forming units (CFU) of live BCG vs. live BCG treated with sodium molybdate.

A–I). PI3P-positive phagosomes were more abundant in the purified organelles in an established in vitro system (5). SapM samples with molybdate (Fig. 4J). A temporal analysis by 4D was added to the in vitro system with purified streptavidin bead microscopy (Fig. 4K) confirmed that molybdate counteracted phagosomes, and late endosomes were preloaded with the fluid the action of a mycobacterial product affecting PI3P levels on phase marker biotin-horse radish peroxidase (HRP). HRP as- phagosomes. Molybdate did not affect mycobacterial viability sociation with streptavidin beads occurs after membrane fusion (Fig. 4L), excluding the possibility that its action was secondary between phagosomal and endosomal organelles and only upon to bacterial killing. The treatment with molybdate also translated mixing of the luminal content in the two compartments. Regu- into enhanced maturation of mycobacterial phagosomes (Fig. 10, lated organellar fusion between phagosomes and late endosomes which is published as supporting information on the PNAS web is quantified as HRP activity bound to streptavidin beads at the site). We conclude that a mycobacterial product sensitive to the end of reaction (5). Addition of SapM inhibited ATP-dependent phosphatase inhibitor molybdate, most likely SapM, has direct or regulated phagosome–late endosome fusion (Fig. 5A). In con- indirect access in vivo to the PI3P substrate within the phago- trast, addition of purified GST-MptpA and GST-MptpB did not somal membrane. inhibit fusion relative to the control with GST alone (data not shown). The fusion depended on PI3P generation, because it was M. tuberculosis PI3P Phosphatase SapM Inhibits PI3P-Dependent affected by the PI3K inhibitor wortmannin (Fig. 5 Inset). Thus, Phagosome–Late Endosome Fusion in Vitro. We next examined the PI3P phosphatase activity secreted by M. tuberculosis has an CELL BIOLOGY whether SapM affects phagosome–late endosome fusion using inhibitory effect on phagosome–lysosome fusion.

Vergne et al. PNAS ͉ March 15, 2005 ͉ vol. 102 ͉ no. 11 ͉ 4037 Downloaded by guest on October 2, 2021 membrane leaflet. It is possible that SapM is exported to the cytosolic side, or alternatively there may be a mechanism for PI3P presentation to SapM remaining on the luminal side. Either way, our data with molybdate, a membrane-impermeant (39, 40) inhibitor of SapM, indicate that, in vivo, SapM gains access to phagosomal PI3P. In the previous and present work, we and others have pinpointed the key membrane-trafficking processes targeted by mycobacteria (11, 14, 26). The mechanism of M. tuberculosis phagosome– lysosome fusion arrest seems to be at least a two-prong process converging upon PI3P. In earlier work (12, 24), we have identified that M. tuberculosis glycolipid, lipoarabinomannan, interferes with Ca2ϩ rise and recruitment͞activation of PI3K hVPS34 (24). The inhibition of Ca2ϩ rise (26) is important but not sufficient to maintain the maturation block, because M. tuberculosis has to preserve a PI3P-negative status during its long-term residence in infected macrophages. We have now uncovered the second com- Fig. 5. M. tuberculosis-secreted PI3P phosphatase SapM inhibits phago- ponent of this double-latch mechanism, in the form of a mycobac- some–late endosome fusion in vitro. The standard phagosome–late endo- terial heat-sensitive enzymatic activity that removes PI3P from some in vitro fusion reaction was carried as described (5), in the presence of 32 membranes. This finding explains the difference between heat- ␮g͞ml of puriﬁed SapM (Mt SapM). (Inset) Fusion reaction in the presence of killed and live mycobacteria, because phagosomes containing heat- 100 nM wortmannin (Wm). Ctrl, untreated control. Error bars represent SEM. M tuber- Ͻ ϭ inactivated bacilli, in sharp contrast to those with viable . *, P 0.05, n 3. culosis, remain strongly PI3P-positive and mature into the phagolysosome. Conclusion The critical role of PI3P in elimination of intracellular mycobacteria was recently underscored in the context of the PI3P-dependent In this study, we have uncovered the principal difference between process of autophagy, which can be induced in infected macro- live and dead intracellular mycobacteria. Live mycobacteria remove phages by activation with the protective cytokine IFN-␥, overcom- the PI3P from the phagosomal membrane, thus precluding organel- ing the M. tuberculosis phagolysosome biogenesis block (23). Our lar maturation. PI3P provides a marquee membrane tagging signal, identification of the M. tuberculosis enzymatic entity responsible for earmarking phagosomes for maturation (11, 16). Dead mycobac- the PI3P removal from phagosomes as the previously characterized teria have no ability to remove PI3P from their phagosomal mycobacterial acid phosphatase SapM, provides a new target for membrane and mature down the phagolysosome biogenesis pathway. Live mycobacteria secrete a PI3P phosphatase activity that drug and vaccine development. Such interventions, directed at inhibits fusion between phagosomes and late endosomal͞lysosomal preventing the establishment of intracellular M. tuberculosis, may organelles. The secreted M. tuberculosis PI3P phosphatase is sen- disrupt the vicious cycle of tuberculosis persistence and propagation sitive to heat inactivation, and correlates fully with the removal of in human populations. PI3P from phagosomes harboring live but not dead mycobacteria. The M. tuberculosis PI3P activity was narrowed down to SapM, an We thank E. Roberts for suggestions regarding chloramphenicol treatment; M. Clague, J. Dixon, H. Stenmark, and M. Yaffe for plasmid acid phosphatase previously studied by Saleh and Belisle (30). constructs; and A. Koul for purified MPtpA and MPtpB. M. tuberculosis Belisle and colleagues have shown that SapM is secreted by culture filtrate protein was prepared with the support of National mycobacteria, a finding further extended in the present study. It Institute of Allergy and Infectious Diseases Contract NO1 AI-75320 remains to be determined how SapM, once secreted into the titled ‘‘Tuberculosis Research Materials and Vaccine Testing.’’ This phagosomal lumen, gains access to PI3P within the cytofacial work was supported by National Institutes of Health Grant AI45148.

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