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...

SummaryListeriolysin O (LLO) is a thiol-activated cytolysin secreted by Listeria monocytogenes. LLO and phosphatidylinositol phospholipase C are two essential virulence factors, which this bacterium needs to escape from the phagosomal compartment to the cytoplasm. Cathepsin-D specifically cleaves LLO, between the Trp-491 (tryptophan amino acid in three letter nomenclature) and Trp-492 residues of the conserved undecapeptide sequence, ECTGLAWEWWR, in the domain 4 of LLO (D4). Moreover, these residues also correspond to the phagosomal-binding epitope. Cathepsin-D had no effect on phosphatidylinositol phospholipase C. We have observed that cathepsin-D cleaved the related cholesterol-dependent cytolysin pneumolysin at the same undecapeptide sequence between Trp-435 and Trp-436 residues. These studies also revealed an additional cathepsin-D cleavage site in the pneumolysin D4 domain localized in the 361-GDLLLD-366 sequence. These differences might confer a pathogenic advantage to listeriolysin O, increasing its resistance to phagosomal cathepsin-D action by reducing the number of cleavages sites in the D4 domain. Using DLLO/W491A and DLLO/W492A bacterial mutants, we reveal that the Trp-491 residue has an important role linked to cathepsin-D in Listeria innate immunity.

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