Complementary activities of SseJ and SifA regulate dynamics of the Salmonella typhimurium vacuolar membrane

The Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS) of Salmonella typhimurium is required for bacterial replication within host cells. It acts by translocating effector proteins across the membrane of the Salmonella-containing vacuole (SCV). The SifA effector is required to maintain the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella-induced filaments (Sifs), which are tubular extensions of SCVs. We have investigated the role in S. typhimurium virulence of the putative SPI-2 effector genes sifB, srfJ, sseJ and sseI. An S. typhimurium strain carrying a mutation in sseJ was mildly attenuated for systemic virulence in mice, but strains carrying mutations in either srfJ, sseI or sifB had very little or no detectable virulence defect after intraperitoneal inoculation. Expression of SseJ in HeLa cells resulted in the formation of globular membranous compartments (GMCs), the composition of which appears to be similar to that of SCV membranes and Sifs. The formation of GMCs was dependent on the serine residue of the predicted acyltransferase/lipase active site of SseJ. Transiently expressed SseJ also inhibited Sif formation by wild-type bacteria, and was found to associate with Sifs, SCV membranes and simultaneously expressed SifA. Intracellular vacuoles containing sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant, a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating that loss of vacuolar membrane around sifA mutant bacteria requires the action of SseJ. Collectively, these results suggest that the combined action of SseJ and SifA regulate dynamics of the SCV membrane in infected cells.     

Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments

Salmonella typhimurium is a facultative intracellular pathogen that colonizes host cells throughout the course of infection. A unique feature of this pathogen is its ability to enter into (invade) epithelial cells and elongate the vacuole within which it resides into tubular structures called Salmonella-induced filaments (Sifs). In this study we sought to characterize the mechanism of Sif formation by immunofluorescence analysis using subcellular markers. The late endosomal lipid lysobisphosphatidic acid associated in a punctate pattern with the Salmonella-containing vacuole, starting 90 min after infection and increasing thereafter. Lysobisphosphatidic acid-rich vesicles were also found to interact with Sifs, at numerous sites along the tubules. Similarly, cholesterol-rich vesicles were also found in association with intracellular bacteria and Sifs. The lysosomal hydrolase cathepsin D was present in Sifs, both in a punctate pattern and, at later times, predominantly in an uninterrupted linear pattern. Rab7 associated with Sifs and expression of the N125I dominant negative mutant of this GTPase inhibited Sif formation. Transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused swelling and aggregation of lysobisphosphatidic acid-containing compartments, suggesting that this virulence factor directs membrane fusion events involving late endosomes. Our findings demonstrate that Sif formation involves fusion of late endocytic compartments with the Salmonella-containing vacuole, and suggest that SifA modulates this event.     

Dynein-mediated vesicle transport controls intracellular Salmonella replication

Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.     

Salmonella type III effectors PipB and PipB2 are targeted to detergent-resistant microdomains on internal host cell membranes

The intracellular pathogen, Salmonella enterica, translocates type III effectors across its vacuolar membrane into host cells. Herein we describe a new Salmonella effector, PipB2, which has sequence similarity to another type III effector, PipB. In phagocytic cells, PipB2 localizes to the Salmonella-containing vacuole (SCV) and tubular extensions from the SCV, Salmonella-induced filaments (Sifs). We used the specific targeting of PipB2 in macrophages to characterize Sifs in phagocytic cells for the first time. In epithelial cells, PipB2 has a unique localization pattern, localizing to SCVs and Sifs and additionally to vesicles at the periphery of infected cells. We further show that the N-terminal 225-amino-acid residues of PipB2 are sufficient for type III translocation and association with SCVs and Sifs, but not peripheral vesicles. Subcellular fractionation demonstrated that both PipB and PipB2 associate with host cell membranes and resist extraction by high salt, high pH and to a significant extent, non-ionic detergent. Furthermore, PipB and PipB2 are enriched in detergent-resistant microdomains (DRMs), also known as lipid rafts, present on membranes of SCVs and Sifs. The enrichment of Salmonella effectors in DRMs on these intracellular membranes probably permits specific interactions with host cell molecules that are concentrated in these signalling platforms.     

Effects of lysosomal membrane protein depletion on the Salmonella-containing vacuole

Salmonella is an intracellular bacterial pathogen that replicates within a membrane-bound vacuole in host cells. The major lysosomal membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are recruited to the Salmonella-containing vacuole as well as Salmonella- associated filaments (Sifs) that emerge from the vacuole. LAMP-1 is a dominant membrane marker for the vacuole and Sifs. Its colocalization with both is dependent on a major secreted bacterial virulence protein, SifA. Here, we show that SifA is required for the recruitment of LAMP-2 and can be used as a second independent marker for both the bacterial vacuolar membrane and Sifs. Further, RNAi studies revealed that in LAMP-1 depleted cells, the bacteria remain membrane bound as measured by their association with LAMP-2 protein. In contrast, LAMP-2 depletion increased the amount of LAMP-1 free bacteria. Together, the data suggests that despite its abundance, LAMP-1 is not essential, but LAMP-2 may be partially important for the Salmonella-containing vacuolar membrane.     

A Salmonella typhimurium effector protein SifA is modified by host cell prenylation and S-acylation machinery

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.     

Salmonella SPI1 effector SipA persists after entry and cooperates with a SPI2 effector to regulate phagosome maturation and intracellular replication

Salmonellae employ two type III secretion systems (T3SSs), SPI1 and SPI2, to deliver virulence effectors into mammalian cells. SPI1 effectors, including actin-binding SipA, trigger initial bacterial uptake, whereas SPI2 effectors promote subsequent replication within customized Salmonella-containing vacuoles (SCVs). SCVs sequester actin filaments and subvert microtubule-dependent motors to migrate to the perinuclear region. We demonstrate that SipA delivery continues after Salmonella internalization, with dosage being restricted by host-mediated degradation. SipA is exposed on the cytoplasmic face of the SCV, from where it stimulates bacterial replication in both nonphagocytic cells and macrophages. Although SipA is sufficient to target and redistribute late endosomes, during infection it cooperates with the SPI2 effector SifA to modulate SCV morphology and ensure perinuclear positioning. Our findings define an unexpected additional function for SipA postentry and reveal precise intracellular communication between effectors deployed by distinct T3SSs underlying SCV biogenesis.     

SifA permits survival and replication of Salmonella typhimurium in murine macrophages

SifA was originally identified as a virulence factor required for formation of Salmonella -induced filaments (Sifs), elongated tubules rich in lysosomal glycoproteins that extend from the Salmonella -containing vacuole in infected epithelial cells. Here, we demonstrate that deletion mutants of ssaR , a component of the SPI-2 type III secretion system, do not form Sifs in HeLa epithelial cells. This suggests that SifA is a translocated effector of this system, acting within host cells to form Sifs. In support of this hypothesis, transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused extensive vacuolation of LAMP-1-positive compartments. Filamentous tubules that closely resembled Sifs were also observed in transfected cells, demonstrating that SifA is sufficient to initiate alteration of host cell endosomal structures. Δ sifA mutants were impaired in their ability to survive/replicate in RAW 264.7 murine macrophages, a phenotype similar to ssaR mutants. Our findings suggest that SifA is an effector of the SPI-2 type III secretion system and allows colonization of murine macrophages, the host niche exploited during systemic phases of disease in these animals. A family of SifA-related proteins and their importance to Salmonella pathogenesis is also discussed.     

A role for Rab7 in the movement of secretory granules in cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP-bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over-expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus-end motor, dynein.     

The SPI-2 type III secretion system restricts motility of Salmonella-containing vacuoles

Intracellular replication of Salmonella enterica occurs in membrane-bound compartments, called Salmonella-containing vacuoles (SCVs). Following invasion of epithelial cells, most SCVs migrate to a perinuclear region and replicate in close association with the Golgi network. The association of SCVs with the Golgi is dependent on the Salmonella-pathogenicity island-2 (SPI-2) type III secretion system (T3SS) effectors SseG, SseF and SifA. However, little is known about the dynamics of SCV movement. Here, we show that in epithelial cells, 2 h were required for migration of the majority of SCVs to within 5 microm from the microtubule organizing centre (MTOC), which is located in the same subcellular region as the Golgi network. This initial SCV migration was saltatory, bidirectional and microtubule-dependent. An intact Golgi, SseG and SPI-2 T3SS were dispensable for SCV migration to the MTOC, but were essential for maintenance of SCVs in that region. Live-cell imaging between 4 and 8 h post invasion revealed that the majority of wild-type SCVs displaced less than 2 microm in 20 min from their initial starting positions. In contrast, between 6 and 8 h post invasion the majority of vacuoles containing sseG, sseF or ssaV mutant bacteria displaced more than 2 microm in 20 min from their initial starting positions, with some undergoing large and dramatic movements. Further analysis of the movement of SCVs revealed that large displacements were a result of increased SCV speed rather than a change in their directionality, and that SseG influences SCV motility by restricting vacuole speed within the MTOC/Golgi region. SseG might function by tethering SCVs to Golgi-associated molecules, or by controlling microtubule motors, for example by inhibiting kinesin recruitment or promoting dynein recruitment.     

Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor betalll spectrin

The small GTPase Rab7 controls late endocytic transport by the minus end-directed motor protein complex dynein-dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein-related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP-Rab7-ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150(Glued), which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150(Glued) recruitment by Rab7-RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as betaIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150(Glued) dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7-RILP is transferred by ORP1L to betaIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with betaIII spectrin, which requires the activities of Rab7-RILP and ORP1L.     

A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome-lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.     

The Rab7 effector protein RILP controls lysosomal transport by inducing the recruitment of dynein-dynactin motors.

Many intracellular compartments, including MHC class II-containing lysosomes, melanosomes, and phagosomes, move along microtubules in a bidirectional manner and in a stop-and-go fashion due to the alternating activities of a plus-end directed kinesin motor and a minus-end directed dynein-dynactin motor. It is largely unclear how motor proteins are targeted specifically to different compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments after activation, which makes Rab GTPases ideal candidates for controlling motor protein binding to specific membranes. We and others [7] have identified a protein, called RILP (for Rab7-interacting lysosomal protein), that interacts with active Rab7 on late endosomes and lysosomes. Here we show that RILP prevents further cycling of Rab7. RILP expression induces the recruitment of functional dynein-dynactin motor complexes to Rab7-containing late endosomes and lysosomes. Consequently, these compartments are transported by these motors toward the minus end of microtubules, effectively inhibiting their transport toward the cell periphery. This signaling cascade may be responsible for timed and selective dynein motor recruitment onto late endosomes and lysosomes.     

The Rab Interacting Lysosomal Protein (RILP) Homology Domain Functions as a Novel Effector Domain for Small GTPase Rab36: Rab36 REGULATES RETROGRADE MELANOSOME TRANSPORT IN MELANOCYTES

Small GTPase Rab functions as a molecular switch that drives membrane trafficking through specific interaction with its effector molecule. Thus, identification of its specific effector domain is crucial to revealing the molecular mechanism that underlies Rab-mediated membrane trafficking. Because of the large numbers of Rab isoforms in higher eukaryotes, however, the effector domains of most of the vertebrate- or mammalian-specific Rabs have yet to be determined. In this study we screened for effector molecules of Rab36, a previously uncharacterized Rab isoform that is largely conserved in vertebrates, and we succeeded in identifying nine Rab36-binding proteins, including RILP (Rab interacting lysosomal protein) family members. Sequence comparison revealed that five of nine Rab36-binding proteins, i.e. RILP, RILP-L1, RILP-L2, and JIP3/4, contain a conserved coiled-coil domain. We identified the coiled-coil domain as a RILP homology domain (RHD) and characterized it as a common Rab36-binding site. Site-directed mutagenesis of the RHD of RILP revealed the different contributions by amino acids in the RHD to binding activity toward Rab7 and Rab36. Expression of RILP in melanocytes, but not expression of its Rab36 binding-deficient mutants, induced perinuclear aggregation of melanosomes, and this effect was clearly attenuated by knockdown of endogenous Rab36 protein. Moreover, knockdown of Rab36 in Rab27A-deficient melanocytes, which normally exhibit perinuclear melanosome aggregation because of increased retrograde melanosome transport activity, caused dispersion of melanosomes from the perinucleus to the cell periphery, but knockdown of Rab7 did not. Our findings indicated that Rab36 mediates retrograde melanosome transport in melanocytes through interaction with RILP.     

Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation

Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome‐lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co‐localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA‐Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ‐BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein‐sorting (HOPS) complex hampered the co‐localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway.      

Salmonella maintains the integrity of its intracellular vacuole through the action of SifA

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.     

SifA,a type III secreted effector of Salmonella typhimurium,directs Salmonella-induced filament (Sif) formation along microtubules

A unique feature of Salmonella enterica serovar typhimurium ( S. typhimurium ) is its ability to enter into (invade) epithelial cells and elongate the vacuole it occupies into tubular structures called Salmonella -induced filaments (Sifs). This phenotype is dependent on SifA, a Salmonella virulence factor that requires the SPI-2-encoded type III secretion system for delivery into host cells. Previous attempts to study SifA and other type III secreted proteins have been limited by a lack of suitable reagents. We examined SifA function by expressing SifA with two internal hemagglutinin epitope tags. By employing subcellular fractionation techniques, we determined that translocated SifA was membrane associated in infected HeLa cells. Confocal microscopy revealed that SifA associated with the Salmonella vacuole and with Sifs. Our analysis also revealed that microtubules serve as a scaffold for Sifs, and that SifA colocalizes with microtubules at sites of interaction between lysosomal glycoprotein-containing vesicles and Sifs. Treatment with the microtubule inhibitor nocodazole blocked Sif formation but did not prevent SifA translocation into the Salmonella vacuole. While polymerized actin has been observed on Sifs, this phenotype was transient and did not play a role in promoting or maintaining Sif formation. Our findings demonstrate the essential role of microtubule dynamics in the formation of Sifs and the utility of this epitope tagging strategy for the study of bacterial type III secreted proteins.     

Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells

Salmonella Typhimurium is a facultative intracellular pathogen that causes acute gastroenteritis in man. Intracellular Salmonella survive and replicate within a modified phagosome known as the Salmonella-containing vacuole (SCV). The onset of intracellular replication is accompanied by the appearance of membrane tubules, called Salmonella-induced filaments (Sifs), extending from the SCV. Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized. In this study, we use live cell imaging techniques to define the dynamics of Sif formation in infected epithelial cells. At early time-points, Sifs are simple tubules extending from the surface of SCVs. These tubules are highly dynamic and exhibit bidirectional, microtubule-dependent movement. At the distal ends of individual Sif tubules, furthest from the SCV, a distinct 'leader' domain was often observed. At later times, Sifs develop into highly complex tubular networks that extend throughout the cell and appear less dynamic than nascent Sifs; however, individual tubules continue to display bidirectional dynamics. Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway. Together, these results show that these Salmonella-induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.     

Salmonella typhimurium SifA effector protein requires its membrane-anchoring C-terminal hexapeptide for its biological function

SifA is a Salmonella typhimurium effector protein that is translocated across the membrane of the Salmonella-containing vacuole by the Salmonella pathogenicity island 2-encoded type III secretion system. SifA is necessary for the formation of Salmonella-induced filaments and for the maintenance of the vacuolar membrane enclosing the pathogen. We have investigated the role of the C-terminal hexapeptide of SifA as a potential site for membrane anchoring. An S. typhimurium strain carrying a deletion of the sequence encoding this hexapeptide (sifA Delta 6) was found to be attenuated for systemic virulence in mice. In mouse macrophages, sifA Delta 6 mutant bacteria displayed a reduced association with vacuolar markers, similar to that of sifA null mutant bacteria, and exhibited a dramatic replication defect. Expression of SifA in epithelial cells results in the mobilization of lysosomal glycoproteins in large vesicular structures and Sif-like tubules. This process requires the presence of the C-terminal hexapeptide domain of SifA. Ectopic expression of truncated or mutated versions of SifA affecting the C-terminal hexapeptide revealed a strong correlation between the membrane binding capability and the biological activity of the protein. Finally, the eleven C-terminal residues of SifA are shown to be sufficient to target the Aequorea green fluorescent protein to membranes. Altogether, our results indicate that membrane anchoring of SifA requires its C-terminal hexapeptide domain, which is important for the biological function of this bacterial effector.     

Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP

Nascent phagosomes must undergo a series of fusion and fission reactions to acquire the microbicidal properties required for the innate immune response. Here we demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP (Rab7-interacting lysosomal protein), which in turn bridges phagosomes with dynein-dynactin, a microtubule-associated motor complex. The motors not only displace phagosomes in the centripetal direction but, strikingly, promote the extension of phagosomal tubules toward late endocytic compartments. Fusion of tubules with these organelles was documented by fluorescence and electron microscopy. Tubule extension and fusion with late endosomes and/or lysosomes were prevented by expression of a truncated form of RILP lacking the dynein-dynactin-recruiting domain. We conclude that full maturation of phagosomes requires the retrograde emission of tubular extensions, which are generated by activation of Rab7, recruitment of RILP, and consequent association of phagosomes with microtubule-associated motors.