Mechanism of Phagolysosome Biogenesis Block by Viable Mycobacterium Tuberculosis
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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 phago- some maturation (11, 16). However, the status of PI3P on phago- somal 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, immunofluores- cence 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)