Phagosomes induced by cytokines function as anti-Listeria vaccines: novel role for functional compartmentalization of STAT-1 protein and cathepsin-D.

Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91-99)/CD8(+)- and LLO(189-201)/CD4(+)-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis.

LIMP-2 links late phagosomal trafficking with the onset of the innate immune response to Listeria monocytogenes: a role in macrophage activation.

The innate immune response to Listeria monocytogenes depends on phagosomal bacterial degradation by macrophages. Here, we describe the role of LIMP-2, a lysosomal type III transmembrane glycoprotein and scavenger-like protein, in Listeria phagocytosis. LIMP-2-deficient mice display a macrophage-related defect in Listeria innate immunity. They produce less acute phase pro-inflammatory cytokines/chemokines, MCP-1, TNF-α, and IL-6 but normal levels of IL-12, IL-10, and IFN-γ and a 25-fold increase in susceptibility to Listeria infection. This macrophage defect results in a low listericidal potential, poor response to TNF-α activation signals, impaired phago-lysosome transformation into antigen-processing compartments, and uncontrolled LM cytosolic growth that fails to induce normal levels of acute phase pro-inflammatory cytokines. LIMP-2 transfection of CHO cells confirmed that LIMP-2 participates in the degradation of Listeria within phagosomes, controls the late endosomal/lysosomal fusion machinery, and is linked to the activation of Rab5a. Therefore, the role of LIMP-2 appears to be connected to the TNF-α-dependent and early activation of Listeria macrophages through internal signals linking the regulation of late trafficking events with the onset of the innate Listeria immune response.

The innate immunity role of cathepsin-D is linked to Trp-491 and Trp-492 residues of listeriolysin O.

Listeriolysin 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 ΔLLO/W491A and ΔLLO/W492A bacterial mutants, we reveal that the Trp-491 residue has an important role linked to cathepsin-D in Listeria innate immunity.

Characterization of a Listeria monocytogenes protein interfering with Rab5a.

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.

Cutting edge: a novel nonoxidative phagosomal mechanism exerted by cathepsin-D controls Listeria monocytogenes intracellular growth.

Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells is a key issue in understanding the pathogenesis of this food-borne pathogen, which can cause diseases ranging from gastroenteritis to meningitis and abortion. In this study, we show that the lysosomal aspartyl-protease cathepsin-D (Ctsd) is of considerable importance for nonoxidative listericidal defense mechanisms. We observed enhanced susceptibility to L. monocytogenes infection of fibroblasts and bone-marrow macrophages and increased intraphagosomal viability of bacteria in fibroblasts isolated from Ctsd-deficient mice compared with wild type. These findings are further supported by prolonged survival of L. monocytogenes in Ctsd-deficient mice after infection. Transient transfection of Ctsd in wild-type cells was sufficient to revert these wild-type phagosomes back to microbicidal compartments. Based on infection experiments with mutant bacteria, in vitro degradation, and immunoprecipitation experiments, we suggest that a major target of cathepsin D is the main virulence factor listeriolysin O.