Cell-Free Fusion of Bacteria-Containing Phagosomes with Endocytic Compartments

Cell-free fusion of bacteria-containing phagosomes with endocytic compartments Ulrike Becken, Andreas Jeschke, Katharina Veltman1, and Albert Haas2 Cell Biology Institute, University of Bonn, Ulrich-Haberland-Strasse 61a, 53121 Bonn, Germany Edited by Reinhard Jahn, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, and accepted by the Editorial Board October 4, 2010 (received for review June 2, 2010) Uptake of microorganisms by professional phagocytic cells leads to duced fusion of SCPs with lysosomes compared to phagosomes formation of a new subcellular compartment, the phagosome, containing inert particles (10–12), others did not (13). Fusogeni- which matures by sequential fusion with early and late endocytic city of SCPs with other endocytic organelles in macrophages has compartments, resulting in oxidative and nonoxidative killing of been investigated little (10). the enclosed microbe. Few tools are available to study membrane To truly understand the (dys)function of phagosome matura- fusion between phagocytic and late endocytic compartments in tion at a molecular level, the fusion of endocytic with microbe- general and with pathogen-containing phagosomes in particular. containing phagocytic organelles ought to be investigated in We have developed and applied a fluorescence microscopy assay reconstituted systems, yet most published assays used bead- to study fusion of microbe-containing phagosomes with different- containing but not microbe-containing phagosomes (3, 5) and aged endocytic compartments in vitro. This revealed that fusion were limited to fusion of early phagosomes with early endocytic of phagosomes containing nonpathogenic Escherichia coli with organelles (14, 15). Fusion of lysosomes with microbe-containing lysosomes requires Rab7 and SNARE proteins but not organelle phagosomes has been reconstituted only in permeabilized macro- acidification. In vitro fusion experiments with phagosomes con- phages (16). Here, we present a generally applicable in vitro taining pathogenic Salmonella enterica serovar Typhimurium assay to quantify fusion of phagosomes containing pathogenic indicated that reduced fusion of these phagosomes with early or harmless microorganisms or inert particles with endocytic and late endocytic compartments was independent of endosome organelles of different maturation stages. and cytosol sources and, hence, a consequence of altered phagosome quality. Results Maturation of Phagosomes Containing Heat-Killed Escherichia coli latex beads ∣ phagocytosis ∣ macrophage ∣ phagolysosome ∣ vacuole DH5α or Latex Beads in Vivo. Kinetic analysis of phagolysosome formation in J774E macrophages using fluorescently labeled, hagocytosis is the receptor-mediated ingestion of large heat-killed, IgG-opsonized E. coli showed that colocalization of particles, usually by professional phagocytes, i.e., neutrophils, bacteria with preloaded lysosomal dye was almost quantitative P “ dendritic cells, monocytes, or macrophages, resulting in forma- after 30 min at 37 °C (Fig. S1 and SI Text). Therefore 30-min ” tion of a new cytoplasmic compartment, the phagosome (1). phagosomes (phagolysosomes) were used in fusion experiments. The development of a phagosome into a fully degradative Phagosomes containing IgG-coupled latex beads prepared after phagolysosome is temporally and spatially ordered in that the 30-min infection and 60-min chase were enriched in LAMP1 but Æ 4 n ¼ 3 newly formed “early” phagosome matures vectorially into a “late” not TfR (Fig. S2A). At this time point 84% (SD , )of phagosome after fusion with late endosomes and finally into a beads colocalized with preloaded lysosomal dye. phagolysosome by fusion with lysosomes (1). This process is ac- companied by loss of early endocytic markers [e.g., transferrin Preparation of Endocytic Vesicles. Using pulse/chase protocols for receptor (TfR), Rab5] and acquisition of late endocytic markers ferrofluid (10-nm paramagnetic particles) (17), different-aged [e.g., lysosome associated membrane protein 1 (LAMP1), cathe- endocytic compartments were purified from postnuclear superna- psin D, Rab7] (1, 2). Fusion yield (fusion events per time period tants (PNSs) and analyzed biochemically (Fig. 1 and SI Text). 100∕00 per organelle) generally decreases with endosome or phagosome Compared with PNSs, (pulse/chase) endosomes were age (2, 3). Rab GTPases and SNARE proteins are key regulators strongly enriched in early endocytic TfR and Syntaxin 13 (Stx13), of all vesicle fusion events in the endocytic pathway. Rab proteins, yet contained little late endocytic LAMP1 or mature cathepsin D. in concert with tethering factors, specifically bridge membranes Both Rab5 and Rab7 were enriched and, on a single-organelle to be fused (4) with Rab5 supporting early (3, 5) and Rab7 level, 50% of endosomes were positive for both Rabs (Fig. S3 100∕150 supporting late (6) fusion events, but additional Rabs may be and SI Text). endosomes were also enriched in TfR involved (7). SNARE proteins on both partner membranes and Stx13, but showed more late endosome characteristics, such are downstream effectors of Rab proteins and drive membrane as loss of Rab5, with only modest enrichment of lysosomal BSA β fusion by formation of intermembrane quarternary SNARE com- rhodamine and lysosomal acid -galactosidase activity. Lyso- 50∕1200 plexes (8) composed of one R-SNARE helix from one partner somes ( ) were strongly enriched in LAMP1, cathepsin β membrane and Qa-, Qb-, and Qc-SNARE helices from the other. D, acid -galactosidase activity, and BSA rhodamine (Fig. 1 A Following membrane fusion, quarternary SNARE complexes and B) and lacked early endocytic markers. are disassembled under ATP hydrolysis by the N-ethylmaleimide sensitive factor (NSF) reforming activated SNAREs (8). Author contributions: U.B. and A.H. designed research; U.B., A.J., and K.V. performed Several intracellular pathogens interfere with maturation of research; U.B. and A.H. analyzed data; and U.B. and A.H. wrote the paper. their phagosomes into fully degradative compartments, e.g., by The authors declare no conflict of interest. disconnection of the phagosome from the endocytic pathway This article is a PNAS Direct Submission. R.J. is a guest editor invited by the Editorial Board. (e.g., Legionella pneumophilae) or by the arrest of phagosome ma- 1Present address: Institute for Infectiology, ZMBE, University of Münster, turation at a prelysosome stage (e.g., Mycobacterium tuberculosis) Von-Esmarch-Strasse 56, 48149 Muenster, Germany. (1). Macrophage phagosomes containing Salmonella enterica 2To whom correspondence should be addressed. E-mail: [email protected]. (SCP) acidify (9) and acquire LAMP1 but exclude mannose This article contains supporting information online at www.pnas.org/lookup/suppl/ 6-phosphate receptor (10). Whereas several groups observed re- doi:10.1073/pnas.1007295107/-/DCSupplemental. 20726–20731 ∣ PNAS ∣ November 30, 2010 ∣ vol. 107 ∣ no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1007295107 Downloaded by guest on October 4, 2021 (18–23). Neither bafilomycin A1, a specific inhibitor of the vATPase, nor nigericin, a Kþ∕Hþ antiporter and proton gradient uncoupler, inhibited ECP-lysosome fusion in vitro, although either drug effectively collapsed lysosome pH gradients in vitro (SI Text and Fig. S5). Nocodazole inhibited ECP-lysosome fusion in vitro to a minor degree (Fig. 3B). In vitro fusion of pure phagosomes containing inert latex beads (LBPs) with lysosomes (Fig. 3 and Fig. S2) proceeded with the same kinetics as ECP-lysosome fusion (Fig. 3A), was also dependent on physiological temperature and ATP, and was inhibited by NEM and RabGDI (Fig. 3C). To directly test whether membrane fusion and not possibly membrane attachment was quantified, we performed a micro- scopic Foerster resonance energy transfer (FRET) analysis, which yields a signal only after vesicle content mixing but not during vesicle attachment (24, 25). In samples containing an Fig. 1. Preparation of endocytic vesicles. Macrophages were incubated ATP-depleting system or an inhibitory soluble Q-SNARE with ferrofluid and magnetic compartments were isolated from a PNS. mixture (see below), percentages of bacteria with mean (A) Western blot analysis of PNS and purified endocytic compartments. Fifty > 0 μ μ FRET intensities and percentages of colocalization were re- or 10 g protein of PNS, 10 g protein for each purified fraction were added. duced by the same degree (Fig. 3D), although membrane attach- Pulse/chase times for ferrofluid are indicated. Arrowheads indicate the posi- tions of cathepsin D (Cat D) precursor (p), intermediate (i), and mature (m) ment should have occurred normally (26). forms. (B) Quantification of specific lysosomal acid β-galactosidase activities (U∕mg) and of fluorescence (FU) of lysosomal BSA rhodamine. Shown are means and standard deviations of three independent experiments. Characterization of Phagosome–Lysosome Fusion in Vitro. Cell-free fusion of E. coli-containing phagolysosomes (ECPs) with lysosomes (Fig. 2) progressed quickly and reached a plateau after approximately 40 min (Fig. 3A). In vitro fusion required physiological temperature and ATP, it was partly dependent on cytosol, was inhibited by the fast Ca2þ chelator 1,2-bis(2-aminophenoxy) ethane-N′,N′,N′,N′-tetraacetate (BAPTA), the NSF inhibitor N-ethylmaleimide (NEM), and by recombinant Rab guanosine nucleotide dissociation inhibitor (RabGDI), which can extract Rab(GDP) proteins from membranes (Fig. 3B). Fusion was sensitive to the calmodulin

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