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. BIOCHEMISTRY 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 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.

www.pnas.org/cgi/doi/10.1073/pnas.1007295107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 23, 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 microscopic 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 inhibitors trifluoperazine and W7, but not to W5, which has lower affinity for calmodulin (Fig. S4). These data agree with results from other in vitro fusion reactions

Fig. 3. In vitro fusion of ECPs or LBPs with lysosomes. Fusion rates under standard conditions (control) were set as 100%. This translates to 3–12% of the bacteria or latex beads being positive for the lysosomal fluor. Means and standard deviations of ≥3 independent experiment are shown. p < 0.05, p < 0.01 compared to control. (A) Time course of phagosome–lysosome fusion. Fusion by 75 min (ECPs) or by 60 min (LBPs) was set as 100%. Fusion reactions contained ECPs (open circles, error bars downward) or LBPs (closed Fig. 2. Assay for cell-free phagosome–endosome fusion. (A) Schematic re- squares, error bars upward). (B and C) Compounds added at the beginning of presentation of the cell-free phagosome–endosome fusion assay. See text the fusion reaction: 4 mM NEM, 10 mM BAPTA, 0.5 mg∕mL RabGDI, 40 nM for details. (B) Micrograph showing a representative image section of an bafilomycin A1,1μM nigericin, 20 μM nocodazole. Reactions contained ECPs in vitro ECP-lysosome fusion reaction prepared for fluorescence microscopy. (B)orLBPs(C). (D–F) Reactions contained ECPs. (D) Colocalization of bacteria In the overlay, phagosomes containing E. coli are shown in green, lysosomes with lysosomal BSA rhodamine and percentage of bacteria with mean in red. Enlargements show bacteria not colocalizing with lysosomal fluor FRET intensity > 0 were calculated and expressed in percent of the respective (Upper) or colocalizing with lysosomal fluor (Lower). (Right) False color- control. One hundred percent (SD 73, n ¼ 7) of colocalizing bacteria coded (open arrowhead) images of FRET intensity for each pixel (black: showed mean FRET intensity > 0.(E) Affinity purified anti-Rab7 or (F) GST- low intensity; white: high intensity). Bar: 2 μM. Rab7 WT or GST-Rab7T22N were added at the indicated final concentrations.

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1007295107 Becken et al. Downloaded by guest on September 23, 2021 Phagosome–Lysosome Fusion Is Regulated by Rab7 and SNARE Pro- as the suspected cognate R-SNAREs (31–33) prevented fusion teins. Rab7 was a prime candidate for a phagosome–lysosome fu- inhibition (Fig. 4A). sion regulator (6). An affinity purified antibody against Rab7 To analyze whether inhibition of phagosome–lysosome fusion (Fig. 3E) and GDP-locked, dominant-negative Rab7T22N-GST in vitro by solSNARE combinations reflected SNARE specificity, but not wild-type Rab7-GST (27, 28) (Fig. 3F) significantly inhib- we replaced the late endocytic Qa-SNARE Stx7 by an early ited ECP-lysosome fusion in vitro in a concentration-dependent endocytic (Stx13) or a neuronal Qa-SNARE (Stx1A). Preincu- manner. bated solStx13/Stx8/Vti1b inhibited phagosome–lysosome fusion Few studies were published on the involvement of specific as efficiently as preincubated solStx7/Stx8/Vti1b, whereas prein- SNARE proteins in phagosome maturation (29), and none of cubated solStx1A/Stx8/Vti1b inhibited fusion less, yet signifi- them analyzed single maturation steps using in vitro fusion ex- cantly (Fig. 4A). To analyze whether this inhibition pattern periments. Late endocytic VAMP8, VAMP7, Vti1b, Stx7, and paralleled the capability of solSNAREs to form quarternary Stx8 but not early endocytic Stx13 were enriched in the lysosome SNARE complexes during preincubation, we tested for SDS re- fraction, and ER (calnexin) and mitochondrium (mtHsp70) pro- sistance of four-helix SNARE bundles (34) (SI Text). As expected teins were virtually absent (Fig. 4C), arguing for high organelle from fusion results and from ref. 34, solStx7/Stx8/Vti1b/VAMP7 purity and validating the SNARE enrichment data. To directly and solStx7/Stx8/Vti1b/VAMP8 produced SDS resistant com- analyze SNARE participation in ECP-lysosome fusion, we used plexes (Fig. 4B). These were absent when the protein mixtures recombinant cytosolic domains of SNARE proteins (solSNAREs) had been heated in the sample buffer, which separates four-helix (21, 26, 30). Lacking transmembrane domains, they are not able complexes. SolStx13/Stx8/Vti1b/VAMP8 and solStx13/Stx8/ to support fusion reactions, yet can interact with membrane Vti1b/VAMP7 could also form SDS resistant complexes. Stx1A bound SNAREs. Single solSNAREs did not inhibit fusion replaced Stx7 functionally only in combination with VAMP7 (Fig. 4A). SolStx7, solStx8, and solVti1b, added together at the but not with VAMP8, in agreement with previous data (34). beginning of the fusion reaction, inhibited neither, but they did when preincubated together on ice (Fig. 4A). Preincubation of In Vivo Maturation of SCPs. As it was not clear whether SCPs fuse these solQ-SNAREs together with solVAMP8 or solVAMP7 with lysosomes in macrophages, we quantified colocalization of P P phagosomes containing heat-killed ( hk) or viable ( live) station- ary-phase S. enterica with gold-rhodamine labeled lysosomes in J774E macrophages (SI Text and Fig. S6A). After 10 min of

∼5% P P BIOCHEMISTRY infection, of live or hk colocalized with the lysosomal ∼90% P ∼30% P dye, at 40 min of hk, but only of live had fused with lysosomes. This difference remained unchanged during 145 min P (Fig. S6A) and documents the maturation defect of live in J774E cells. Fluorescent labeling of Salmonella prior to infection did P P not alter colocalization of live and hk with the lysosomal dye ∼75% P ∼55% P (Fig. S6B). At 55 min of live and of hk were positive for Rab7-GFP; colocalization with Rab5-GFP was ∼20% for both P ∼70% types of phagosomes. Approximately 80% of live and of P hk colocalized with LAMP1 at this time (Fig. S6C).

In Vitro Fusion of Early and Late SCPs with Endocytic Compartments of Different Maturation Stages. As phagosome maturation was affected by viable Salmonella, we tested whether fusion inhibition can be recapitulated in vitro. We extended the in vitro fusion assay to include the examination of fusogenicity of SCPs with P P earlier endocytic compartments. Different-aged live or hk and different-aged endosomes or lysosomes were incubated either under standard conditions or in the presence of an ATP-depleting system. Fusion rates of different phagosomes with endocytic compartments of the same maturation stage were expressed as percent of the highest colocalization value (Fig. 5 and representative micrographs in Fig. S7). Fusion of early endosomes with early P P live or early hk was significantly more pronounced than fusion of early endosomes with late phagosomes (Fig. 5A). Conversely, P the fusion rate of early hk with late endosomes or lysosomes was P significantly lower than the fusion rate of late hk with the same endocytic organelles (Fig. 5 B and C). In contrast, fusion compe- P Fig. 4. SNARE proteins in cell-free ECP-lysosome fusion. (A) Cell-free fusion tences of early or late live with late endosomes or lysosomes, of ECPs with lysosomes was carried out in the presence of single solSNAREs or respectively, were not significantly different (Fig. 5 B and C). combinations of solSNARES as indicated. Preincubations of solSNAREs for For all endocytic compartments tested, the fusion rate with P P 90 min on ice were done (pre) where indicated or solSNAREs were added hk was higher than with live. directly at the start of the fusion reaction (no pre). Data are means and standard deviations of ≥3 independent experiments. p < 0.05, p < 0.01 Discussion compared to control. (B) Combinations of purified recombinant solSNAREs In this study, we developed and used a cell-free assay to analyze were mixed as indicated in equimolar ratios (12 μM each) and incubated fusion of different-aged endocytic compartments with bacteria- for 90 min on ice. Samples were incubated with SDS-sample buffer without containing phagosomes. This microscopic assay for phagosome- boiling and analyzed by Western blotting using an antibody to Stx8. The white arrowhead indicates the running position of solStx8; the black endosome colocalization is conceptually similar to recently arrowhead indicates SDS resistant four-helix bundles. (C) PNS and purified developed assays to study early endosome docking and fusion lysosomes were analyzed for enrichment or depletion of various compart- (21, 26, 35). Fusion yield in positive control samples containing mental markers by Western blotting. different-aged endocytic compartments (2–20%) was well within

Becken et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 23, 2021 inhibition is prevented, when solQa/b/c- and solR-SNAREs form a quaternary complex already during preincubation. In vitro formation of SDS resistant complexes (as in refs. 34, 42, 43) and fusion inhibition by solQa/b/c-SNARE complexes were promiscuous in that they also occurred when late endocytic Stx7 (Qa) was replaced by early endosomal Stx13 (Qa) or neuronal Stx1A (Qa). There is precedence for SNARE promiscuity in the endocytic system, e.g., the functional replacement of Vti1b by other SNAREs in Vti1b knockout mice (44). In contrast, homotypic early endosome fusion in vitro was inhibited only by early endosomal (solStx13/Vti1a/Stx6) but not neuronal (solStx1/SNAP-25) SNARE domains (21). Yet, combinations of solStx7/Vti1b/Stx8 or solStx13/Vti1b/Stx8 inhibited phago- – Fig. 5. In vitro fusion of Salmonella-containing phagosomes with different- some lysosome fusion more efficiently compared to solStx1A/ aged endocytic compartments. In vitro fusion experiments were carried Vti1b/Stx8. As the first two SNARE groups form SDS resistant

out with early (10 min) or late (55 min) phagosomes containing live (Plive) four-helix bundles with either solVAMP7 or solVAMP8, which or heat-killed (Phk) S. enterica. Fusion rates of phagosomes with endosomes are both present on lysosomes, but the third one only with of different age cannot be directly compared, because fluor contents and solVAMP7, this could indicate that both of these R-SNAREs volume of endocytic organelles likely change with maturation. Therefore, are able to mediate phagosome–lysosome fusion. Inhibition by only fusion reactions with same-stage endocytic compartments were com- solStx1A/Vti1b/Stx8 is then less efficient, because only one of pared. Only phagosomes containing an intact membrane (bacteria were – not antibody accessible) were considered. (A) Fusion reactions contained these R SNAREs is inactivated by this complex. 100∕00 endosomes (early endosomes, EE), (B) 100∕150 endosomes (late endo- Acidification of newly formed phagosomes supports phagoly- somes, LE), or (C) 100∕1200 endosomes (lysosomes, Lys). Data are the means sosome formation (24) and is important for autophagosome– and standard deviations of ≥3 independent experiments. Asterisks or values lysosome fusion (45). It may be important for vesicle budding above open bars without brackets mark p values comparing adjacent open in endosome and phagosome maturation (46, 47), yet, its role and filled bars. p < 0.01;*:p < 0, 05. in fusion of late phagosomes or endosomes with lysosomes has not been analyzed directly. As neither bafilomycin nor nigericin the range of other systems reconstituting fusion of endosomes inhibited phagosome–lysosome fusion, organelle acidification (21, 35, 36) or phagosomes (3, 37). seems not to be required for this specific subreaction of phago- Authentic phagosome-lysosome fusion was observed in vitro, lysosome formation. as reaction kinetics matched those of related cell-free fusion To further understand diversion of phagosome maturation by reactions (5, 21). Further, colocalization was strictly dependent intracellular pathogens, we compared fusion competence of early on a physiological temperature and presence of ATP. Addition and late phagosomes containing live or heat-killed S. enterica with of cytosol increased fusion, but was not absolutely required as different-aged endocytic vesicles. We have shown that in vitro, P has been seen with other in vitro fusion reactions (21, 22), likely early endosomes fuse more avidly with early than with late hk reflecting that fusion-enhancing peripheral membrane proteins and late endosomes and lysosomes are more fusogenic with late P such as NSF or Rab-GTPases are present on isolated membranes. than with early hk as expected. However, some fusion occurred P Like late endosome–lysosome fusion (23), phagosome–lysosome between early hk and late endocytic organelles and, conversely, P fusion was reduced by calmodulin inhibitors, supporting previous late hk and early endosomes. One explanation for this unex- reports on a role for calmodulin in phagosome–lysosome pected finding is heterogenity in the maturation stages of the pha- – P tethering (18). Fusion was also strongly inhibited by NEM and gosomes in that 10 20% of late hk were still positive for early endocytic Rab5, yet negative for lysosomal gold rhodamine. Rab7 RabGDI, reflecting the requirements for NSF and Rab proteins – (19, 20). An important Rab-GTPase in phagosome–lysosome is present on the majority of endosomes that are only 0 10 min fusion was Rab7. Purified Rab proteins were not geranylgerany- old, and this may explain fusion competence between early endosomes and late P in vitro. In vivo, this fusion may not readily lated and, hence, wild-type Rab7 did not promote fusion. Domi- hk occur due to spatial segregation from late phagosomes and nant-negative Rab7 inhibited phagosome–lysosome fusion, as its phagolysosomes. Whether the presence of both Rabs on most function is not fully dependent on the lipid modification (38, 39). early endosomes is a general macrophage characteristic remains Reconstitution of not only ECP- but also LBP-lysosome fusion to be investigated. demonstrates the versatility of the presented assay. Isolated P P Fusion competence of both live and hk with early endosomes LBPs are almost pure allowing in vitro fusion experiments in a P decreased as phagosomes aged. But whereas hk acquired fusion biochemically defined system, albeit with a nonphysiological competence with late endocytic organelles, this was much less the phagosome load. The presented reconstitution system can help P case with live. It has been suggested (48) that late SCPs in macro- to molecularly investigate many of the observations made over phages retain Rab5 and Rab5 effectors, stay fusogenic with early the years with latex bead-fed phagocytes. endosomes, and that this sustained fusion occurs via an uncon- SolSNAREs have previously been used to test SNARE ventional mechanism independent of ATP hydrolysis. We, too, requirements in fusion of endocytic organelles in vitro or in semi- P observed ATP-independent fusion of late live with early endo- intact cells (21, 26, 30). In our study, preincubated combinations somes, yet the fusion extent was much lower than fusion of early of soluble Qa/b/c-SNAREs but not of Qa/b/c/R-SNAREs inhib- P live with early endosomes. And in our study, Rab7-GFP but not ited ECP-lysosome fusion. Because formation of SNARE P Rab5-GFP was associated with the majority of late live. Hence, P four-helix bundles may proceed through Qa/b/c intermediates the strong fusion defect between late live and lysosomes was not (40) and because our solSNAREs form SDS resistant quaternary caused by an early arrest as proposed in ref. 48 but rather by an complexes in vitro, we interpret this as follows: Ternary, three- arrest at a late maturation step. P helix solQa/b/c-SNARE complexes assemble during preincuba- Our data show that the low fusion competence of live with tion on ice at a low rate (34), similar to binary three-helix lysosomes in vivo was reconstituted in vitro and that reduced P SNAP-25/Stx1 complexes (41). The preassembled Q-SNAREs fusion competence of live was not restricted to lysosomes. Such P engage organelle embedded cognate R-SNARE(s) in futile bind- generally reduced fusion competence of live is in line with the ing reactions and so efficiently block membrane fusion. Fusion reported reduced accessibility of SCPs to incoming transferrin

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1007295107 Becken et al. Downloaded by guest on September 23, 2021 and dextran (10). Salmonella effector proteins such as spiC (49), homogenizer. A PNS was prepared at 450 × g for 3 min and centrifuged which are secreted from the phagosome into the cytosol of in- in a fixed angle rotor: When the rotor reached 12;000 × g, the centrifuge fected cells via a type III secretion system, were discussed to was turned off, the supernatant was centrifuged at 12;000 × g for 6 min, be prime candidates for altering maturation of the SCP.However, and the pellet was resuspended in HB∕10 mg∕mL BSA. Preparation of LBPs phagosomes that contained Salmonella generally defective in is described in SI Text. effector protein secretion showed no enhanced fusion with Using pulse/chase protocols, endocytic compartments were loaded with ferrofluid (EMG 508, Ferrotec Europe) and purified from a PNS as in ref. 17 lysosomes (11); hence, the role of effector proteins remains with modifications (SI Text). elusive. Against this background it was revealing that fusion of P live with lysosomes in vitro was diminished with lysosomes from In Vitro Fusion Assay. Standard fusion reactions contained 1.5 μLof10 × salt uninfected cells and did not require cytosol prepared from in- solution (15 mM MgCl2, 1 M KCl), 1.5 μLof10 × ATP regenerating system fected macrophages, which would be a prime source of secreted (SI Text), 0.15 μL 100 mM DTT, 2 mg∕mL cytosol, 2 μL of the endosome or effectors. This suggests that the intraphagosomal bacteria had lysosome preparation and 2 μL of the phagosome preparation. Volumes were turned phagosomes fusion-incompetent before they were iso- adjusted to 15 μL with HB. Each reaction contained lysosomes from 7.5 × 106 lated. Whether reprogramming requires prior bacterial secretion or late or early endosomes from 1.3 × 107 macrophages. Per reaction, bacter- or the presence of certain surface molecules, such as lipopolysac- ia-containing phagosomes from 7.5 × 105 macrophages or LBPs (final concen- charides, remains to be investigated. It has been proposed that tration: A600 ¼ 0.3) were added. In control experiments, cytosol was replaced diminished phagosome–lysosome fusion in vivo may be the result by HB. Recombinant proteins or pharmacologicals were either from a stock in of an impaired capability of SCPs to attach to microtubules (MTs) HB or carrier-containing control samples were included. Fusion experiments and their reduced trafficking (50, 51). Disruption of MTs by were at 37 °C for 60 min and stopped by placing the tubes on ice. To identify – bacteria not surrounded by an intact phagosome membrane, samples with nocodazole had only a minor effect on standard phagosome 5 μ ∕ lysosome fusion in our study, demonstrating that a possible need SCPs were incubated with g mL rabbit anti-Salmonella antibody at 4 °C for 30 min. This labeling was omitted in samples containing ECPs or LBPs. for MTs in phagosome maturation in vivo would be bypassed in Samples were diluted with 1 mL cold HB. Poly-L-lysine coated coverslips were vitro. Hence, inhibition of phagosome movement is not the (only) placed in a 24-well plate, 500 μL of the diluted fusion sample were added per decisive factor in trafficking diversion of SCPs. well, and plates were centrifuged at 1;800 × g for 15 min at 4 °C. Samples were fixed with 2.5% glutardialdehyde and 2% formaldehyde (FA) in HB Material and Methods (samples with ECPs or LBPs) or 3% FA in HB (samples with SCPs and samples Chemicals. Antibodies, plasmids and recombinant proteins, buffers, and pre- for FRET analysis). After rinsing three times with PBS, samples were incubated paration of cytosol are described in SI Text. Cultivation of mammalian and in 1 mg∕mL NaBH4 in PBS (ECPs or LBPs) or 50 mM NH4Cl in HB (SCPs and FRET bacterial cells and their use in infection experiments is described in SI Text. BIOCHEMISTRY analysis) for 30 min. Samples with SCPs were stained with a Cy5-conjugated secondary antibody. Coverslips were mounted in Mowiol. Samples were Fluorescent Labeling of Endocytic Compartments. To label lysosomes for in examined using a Zeiss Axioplan (ECPs or LBPs) or a Zeiss Axio Observer.Z1 vitro fusion experiments with ECPs or LBPs, macrophages were incubated microscope (SCPs and FRET analysis) equipped with 100×/1.4 oil immersion overnight (oN) in complete medium containing 50 μg∕mL BSA rhodamine objectives and appropriate filter sets. Percentages of beads or green bacteria (5 μg∕mL for FRET analysis). Cells were rinsed with PBS, incubated in complete colocalizing with lysosomal BSA rhodamine were determined from 300 medium containing ferrofluid (SI Text) for 5 min, rinsed twice with PBS, and particles each in duplicates. In samples with SCPs, Cy5-labeled bacteria were chased at 37 °C for 2 h. For in vitro fusion experiments with SCPs, ferrofluid excluded from analysis. All coverslips were anonymized. FRET analysis was was incubated oN in BSA rhodamine in PBS and diluted in complete medium resulting in 100 μg∕mL BSA rhodamine and 6 μL∕mL ferrofluid. Macrophages performed according to the principle described by Youvan et al. (52) using were incubated for 10 min at 37 °C with BSA rhodamine/ferrofluid, rinsed Zeiss Axio Vision 4.7.1 software. ATTO488-labeled bacteria were defined twice with PBS, and chased as indicated. For biochemical analysis of purified as regions of interest (R) and mean FRET intensity was calculated for each endocytic compartments (SI Text), lysosomes were labeled with 50 μg∕mL R. Per sample, 200 bacteria were analyzed, and percentages of Rs with mean BSA rhodamine oN, chased for 2 h, and ferrofluid was added. To quantify FRET intensities > 0 were expressed as percent of the positive control. FRET phagosome–lysosome colocalization in vivo, macrophages were incubated intensity for every pixel was displayed in a false color-coded image. in complete medium containing gold rhodamine for 1 h (infection with E. coli:OD520 ¼ 1; infection with S. enterica:OD520 ¼ 0, 4) or containing Statistical Analysis. Means and standard deviations were calculated from 50 μg∕mL BSA rhodamine oN, rinsed thrice with PBS and chased for 3 h independent experiments. Data were analyzed by the two-tailed unpaired (infection with E. coli) or 2 h (infection with S. enterica or latex beads). Student’s t test with significance assumed at p < 0.05 and high significance at p < 0.01. Preparation of Phagosomes and Endocytic Compartments. Infection of macrophages is described in SI Text. Bacteria-containing phagosomes were ACKNOWLEDGMENTS. We thank all colleagues who generously contributed prepared as in ref. 5 with modifications: Cells were sequentially washed plasmids and antibodies. This study was supported by the Deutsche with PBS∕5 mM EDTA and homogenization buffer (HB), resuspended in Forschungsgemeinschaft (SFB 645) with initial support by the Volkswagen HB∕10 mg∕mL BSA∕1 × protease inhibitors, and homogenized in a Dounce Foundation.

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