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...

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...

The use of live Listeria-based vaccines carries serious difficulties when administrated to immunocompromised individuals. However, cellular carriers have the advantage of inducing multivalent innate immunity as well as cell-mediated immune responses, constituting novel and secure vaccine strategies in listeriosis. Here, we compare the protective efficacy of dendritic cells (DCs) and macrophages and their safety. We examined the immune response of these vaccine vectors using two Listeria antigens, listeriolysin O (LLO) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and several epitopes such as the LLO peptides, LLO189−201 and LLO91−99 and the GAPDH peptide, GAPDH1−22. We discarded macrophages as safe vaccine vectors because they show anti-Listeria protection but also high cytotoxicity. DCs loaded with GAPDH1−22 peptide conferred higher protection and security against listeriosis than the widely explored LLO91−99 peptide. Anti-Listeria protection was related to the changes in DC maturation caused by these epitopes, with high production of interleukin-12 as well as significant levels of other Th1 cytokines such as monocyte chemotactic protein-1, tumor necrosis factor-α, and interferon-γ, and with the induction of GAPDH1−22-specific CD4+ and CD8+ immune responses. This is believed to be the first study to explore the use of a novel GAPDH antigen as a potential DC-based vaccine candidate for listeriosis, whose efficiency appears to highlight the relevance of vaccine designs containing multiple CD4+ and CD8+ epitopes.

The mechanisms by which the intracellular pathogen Listeria monocytogenes interacts with the host cell surface remain largely unknown. In this study, we investigated the role of heparan sulfate proteoglycans (HSPG) in listerial infection. Pretreatment of bacteria with heparin or heparan sulfate (HS), but not with other glycosaminoglycans, inhibited attachment and subsequent uptake by IC-21 murine macrophages and CHO epithelial-like cells. Specific removal of HS from target cells with heparinase III significantly impaired listerial adhesion and invasion. Mutant CHO cells deficient in HS synthesis bound and internalized significantly fewer bacteria than wild-type cells did. Pretreatment of target cells with the HS-binding proteins fibronectin and platelet factor 4, or with heparinase III, impaired listerial infectivity only in those cells expressing HS. Moreover, a synthetic peptide corresponding to the HS-binding ligand in Plasmodium falciparum circumsporozoite protein (pepPf1) inhibited listerial attachment to IC-21 and CHO cells. A motif very similar to the HS-binding site of pepPf1 was found in the N-terminal region of ActA, the L. monocytogenes surface protein responsible for actin-based bacterial motility and cell-to-cell spread. In the same region of ActA, several clusters of positively charged amino acids which could function as HS-binding domains were identified. An ActA-deficient mutant was significantly impaired in attachment and entry due to altered HS recognition functions. This work shows that specific interaction with an HSPG receptor present on the surface of both professional and nonprofessional phagocytes is involved in L. monocytogenes cytoadhesion and invasion and strongly suggests that the bacterial surface protein ActA may be a ligand mediating HSPG receptor recognition.