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...

Listeria monocytogenes (LM) modifies the phagocytic compartment by targeting Rab5a function through an unknown mechanism. Inhibition of Rab5a exchange by LM can be considered the main virulence mechanism as it favours viability of the parasite within the phagosome as well as the exclusion of putative listericidal lysosomal proteases such as cathepsin-D. The significance of this survival mechanism is evidenced by the overexpression of Rab5a mutants in CHO cells that promoted GDP exchange on Rab5a and eliminated pathogenic LM. The following mutants showed listericidal effects: Rab5a:Q79L, a constitutively active mutant with accelerated GDP exchange and Rab5a GEF, Vps9, which overactivates the endogenous protein. Clearance of LM from these phagosomes was controlled by the hydrolytic action of cathepsin-D as suggested by the lysosomal protease inhibitor chloroquine, or the cathepsin-D inhibitor, pepstatin A, which caused a reversion of listericidal activity. Moreover, the effects of LM on Rab5a phagocytic function mimics those reported for the GDP locked dominant negative Rab5a mutant, S34N. Transfection of these mutants into CHO cells increased pathogen survival as they showed higher numbers of viable bacteria, complete inhibition of GDP exchange on Rab5a and impairment of the listericidal action probably exerted by cathepsin-D. We cotransfected functional Rab5a GEF into this dominant negative mutant and restored normal LM intraphagosomal viability, Rab5a exchange and listericidal action of cathepsin-D.