ABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Listeria monocytogenes/metabolism , rab5 GTP-Binding Proteins/metabolism , Adenosine Diphosphate Ribose/metabolism , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Clone Cells , Endosomes/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Listeria monocytogenes/genetics , Listeria monocytogenes/pathogenicity , Mice , Molecular Sequence Data , NAD/metabolism , Phagosomes/metabolismABSTRACT
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.
Subject(s)
Cathepsin D/physiology , Listeria monocytogenes/growth & development , Listeria monocytogenes/immunology , Listeriosis/metabolism , Listeriosis/microbiology , Phagosomes/microbiology , Animals , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Cathepsin D/deficiency , Cathepsin D/genetics , Cells, Cultured , Fibroblasts/metabolism , Fibroblasts/microbiology , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Hemolysin Proteins , Immunity, Innate/genetics , Intracellular Fluid/metabolism , Intracellular Fluid/microbiology , Listeriosis/genetics , Macrophages/immunology , Macrophages/microbiology , Mice , Mice, Inbred Strains , Mice, Knockout , Oxidation-Reduction , Phagosomes/metabolism , Virulence Factors/deficiency , Virulence Factors/genetics , Virulence Factors/metabolism , rab5 GTP-Binding Proteins/metabolismABSTRACT
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.
