Survival or destruction of a pathogen following phagocytosis depends, in part, on fusion events between the phagosome and the endosomal or lysosomal compartments. Here we use an in vitro assay to show that phagosome-endosome fusion is regulated by the small GTPase rab5 and that fusion events are influenced by an internalized live organism, Listeria monocytogenes (LM). We compare the in vitro fusion of phagosomes containing heat-killed organisms (dead LM) with that of phagosomes containing a live nonhemolytic mutant (live LMhly-). Unlike the wild-type organism, LMhly- remains trapped inside the phagosome. Phagosome-endosome fusion was reconstituted using biotinylated organisms and endosomes containing horseradish peroxidase conjugated with avidin. With both live LMhly- and dead LM preparations, in vitro phagosome-endosome fusion was time-, temperature-, and cytosol-dependent. Live LMhly- phagosomes exhibited a faster rate of fusion. Fusion in both preparations was regulated by rab5 and possibly by other GTPases. Anti-rab5 antibodies and immunodepletion of cytosolic rab5 inhibited fusion. Addition of glutatione S-transferase-rab5 in the GTP form stimulated phagosome-endosome fusion, whereas addition of a dominant negative mutant of rab5 blocked fusion. Purified live LMhly- phagosomal membranes were enriched in rab5 as revealed by Western blotting, compared with dead LM phagosomes. Fusion of endosomes with dead LM containing phagosomes required ATP and was inhibited by ATP depletion and by N-ethylmaleimide (NEM) and anti-NEM-sensitive factor (NSF) antibodies. Unexpectedly, phagosome-endosome fusion with live LMhly--containing phagosomes was not inhibited by ATP depletion nor by NEM or anti-NSF antibodies. Western blot analysis revealed that live LMhly--containing phagosomes were enriched for membrane-bound NSF, while dead LM containing phagosomes contained low or undetectable quantities. Washing live LMhly--containing phagosomes with 0.5 M KCl removed NSF associated with the membranes and rendered them NEM, ATP, anti-NSF antibody sensitive for fusion. We conclude that rab5 regulates phagosome-endosome fusion and that live microorganisms can up-regulate this process by recruiting rab5 to the membrane.

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH2 and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A “kinase-dead” EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.

Eugenio Carrasco-Marín, deicme@humv.es †These authors contributed equally to this study.Listeria monocytogenes (LM) phagocytic strategy implies recruitment and inhibition of Rab5a. Here, we identify a Listeria protein that binds to Rab5a and is responsible for Rab5a recruitment to phagosomes and impairment of the GDP/GTP exchange activity. This protein was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Listeria (p40 protein, Lmo 2459). The p40 protein was found within the phagosomal membrane. Analysis of the sequence of LM p40 protein revealed two enzymatic domains: the nicotinamide adenine dinucleotide (NAD)-binding domain at the N-terminal and the C-terminal glycolytic domain. The putative ADP-ribosylating ability of this Listeria protein located in the N-terminal domain was examined and showed some similarities to the activity and Rab5a inhibition exerted by Pseudomonas aeruginosa ExoS onto endosome-endosome fusion. Listeria p40 caused Rab5a-specific ADP ribosylation and blocked Rab5a-exchange factor (Vps9) and GDI interaction and function, explaining the inhibition observed in Rab5a-mediated phagosome-endosome fusion. Meanwhile, ExoS impaired Rab5-early endosomal antigen 1 (EEA1) interaction and showed a wider Rab specificity. Listeria GAPDH might be the first intracellular gram-positive enzyme targeted to Rab proteins with ADP-ribosylating ability and a putative novel virulence factor.Key words: ADP-ribosylation, GDP/GTP exchange, glyceraldehyde-3-phosphate dehydrogenase, Listeria, phagocytosis, Rab5a Listeria monocytogenes (LM) is a gram-positive human pathogen that remains for a relatively short time within the phagosomal compartment depending on the cell line. In macrophages, for instance, the average time of bacteria remaining inside the phagosomes is 90 min (1); thereafter, bacteria escape to the cytosol and replicate. During the time LM remains within the phagosome, it modulates the phagosomal composition by targeting Rab5a function and preventing phagosome maturation (2,3). The importance of Rab5 for LM intracellular growth and other infection steps such as entry or vacuolar escape was recently highlighted using small interfering RNA interference technology (4,5). The pathogen produces membrane-active exoproteins within the phagosomes that mediate membrane disruption (6). Intracellular bacteria are able to interfere with vesicle trafficking regulators in order to modify the vesicles in which they reside according to the needs of the specific pathogen. In this regard, there are only a few examples of bacteria whose strategy is target small guanosine triphosphatases (GTPases) exchange activities. For instance, Legionella pneumophila protein RalF functions as a guanine nucleotide exchange factor (GEF) for the ADP ribosylation factor family of small GTPases (7). Salmonella typhimurium SopE protein is another example of an intracellular bacterial factor target small GTPase exchange factor for the Rho/Rab family (8). Recently, our group has described the intracellular trafficking str...

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Control and clearance ofListeria monocytogenes is an intracellular facultative bacterium able to invade phagocytic cells and is responsible for severe pathologies in immunocompromised people, newborns and pregnant women (1). L. monocytogenes entry into the host cell is an active process involving several protein components. After a short phagosomal period (ϳ30 min), L. monocytogenes escapes to the cytosol, avoids intracellular killing, and replicates (reviewed in Ref. 2). The L. monocytogenes survival mechanism involves two steps: (i) live bacteria avoid phagosome maturation by inactivation of the endosomal trafficking regulator Rab5a, which blocks the recruitment of lysosomal proteins to the phagosomes (Lamp-1 and cathepsin-D) (3) and (ii) secretion by L. monocytogenes of listeriolysin and PI-PLC lyses the phagosomal membrane, translocates L. monocytogenes to the cytoplasm, and consequently, allows for L. monocytogenes intracellular survival (4).Control of L. monocytogenes infection and clearance is an interferon-␥ (IFN-␥) 1 -dependent process. IFN-␥ priming of macrophages (MØs) recruited at the inflammatory site triggers their listericidal abilities (5). IFN-␥ signaling modulates the expression and activation of more than 200 proteins (6). However, to date, only a few of these molecules have been shown to exert a direct role in pathogen elimination (7). Among these are (i) IGTP, a GTP-binding protein relevant for Toxoplasma clearance (8) and (ii) Nramp1, a MØ-restricted lysosomal protein involved in Leishmania, Salmonella, and Mycobacterium spp. clearance (9). In addition, IFN-␥ induces the production of reactive oxygen (ROI) and nitrogen (RNI) intermediates with microbicidal activity (10). From this set of molecules, only ROI and RNI have been shown to restrict L. monocytogenes growth (10, 11), while the other two molecules (i.e. IGTP or Nramp1) play no role at all in L. monocytogenes clearance (8, 9).Recently, we have shown that in resting MØs the inhibition of Rab5a synthesis allows for intracellular survival of a listeriolysin-defective L. monocytogenes mutant, that under normal Rab5a levels is unable to grow and fails to escape from the phagosome (12). Furthermore, we have also described that IFN-␥ signaling up-regulates Rab5a function (13). However, at this stage, no correlation between the induction of ROI and RNI by IFN-␥ and the Rab5a function has been established. Here, we show that Rab5a is a key molecule for the IFN-␥ promoted clearance of a pathogenic L. monocytogenes strain at the phagosomal stage. We show that Rab5a, in the GTP form, controls the recruitment of active Rac2 to the transformed L. monocytogenes phagolysosome and the assembly of the phagocyte NADPH oxidase with the production of toxic radicals. These Rab5a-mediated actions compromise Listeria viability within the phagolysosomes and further L. monocytogenes intracellular survival. EXPERIMENTAL PROCEDURESCells and Reagents-J774 cells and proteose peptone-elicited peritoneal MØs from Balb/c mice were cultured in Dulbecco's modified Eag...