Carmen Alvarez-Dominguez

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.

Inhibition of Rab5a exchange activity is a key step for Listeria monocytogenes survival.

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.

A gold glyco-nanoparticle carrying a listeriolysin O peptide and formulated with Advax™ delta inulin adjuvant induces robust T-cell protection against listeria infection

In the search for an effective vaccine against the human pathogen, Listeria monocytogenes (Listeria), gold glyconanoparticles (GNP) loaded with a listeriolysin O peptide LLO91-99 (GNP-LLO) were used to immunise mice, initially using a dendritic cell (DC) vaccine approach, but subsequently using a standard parenteral immunisation approach. To enhance vaccine immunogenicity a novel polysaccharide adjuvant based on delta inulin (Advax™) was also co-formulated with the GNP vaccine. Confirming previous results, DC loaded in vitro with GNP-LLO provided better protection against listeriosis than DC loaded in vitro using free LLO peptide. The immunogenicity of GNP-LLO loaded DC vaccines was further increased by addition of Advax™ adjuvant. However, as DC vaccines are expensive and impracticable for prophylactic use, we next asked whether the same GNP-LLO antigen could be used to directly target DC in vivo. Immunisation of mice with GNP-LLO plus Advax™ adjuvant induced LLO-specific T-cell immunity and protection against Listeria challenge. Protection correlated with an increased frequency of splenic CD4(+) and CD8(+) T cells, NK cells and CD8α(+) DC, and Th1 cytokine production (IL-12, IFN-γ, TNF-α, and MCP-1), post-challenge. Enhanced T-cell epitope recruitment post-challenge was seen in the groups that received Advax™ adjuvant. Immunisation with GNP-LLO91-99 plus Advax™ adjuvant provided equally robust Listeria protection as the best DC vaccine strategy but without the complexity and cost, making this a highly promising strategy for development of a prophylactic vaccine against listeriosis.

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.

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.

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.

Novel nanoparticle vaccines for Listeriosis

In recent years, nanomedicine has transformed many areas of traditional medicine, and enabled fresh insights into the prevention of previously difficult to treat diseases. An example of the transformative power of nanomedicine is a recent nano-vaccine against listeriosis, a serious bacterial infection affecting not only pregnant women and their neonates, but also immune-compromised patients with neoplastic or chronic autoimmune diseases. There is a major unmet need for an effective and safe vaccine against listeriosis, with the challenge that an effective vaccine needs to generate protective T cell immunity, a hitherto difficult to achieve objective. Now utilizing a gold nanoparticle antigen delivery approach together with a novel polysaccharide nanoparticulate adjuvant, an effective T-cell vaccine has been developed that provides robust protection in animal models of listeriosis, raising the hope that one day this nanovaccine technology may protect immune-compromised humans against this serious opportunistic infection.

Cellular vaccines in listeriosis: role of the Listeria antigen GAPDH

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.

Dissociation of innate immune responses in microglia infected with Listeria monocytogenes.

Microglia, the innate immune cells of the brain, plays a central role in cerebral listeriosis. Here, we present evidence that microglia control Listeria infection differently than macrophages. Infection of primary microglial cultures and murine cell lines with Listeria resulted in a dual function of the two gene expression programmes involved in early and late immune responses in macrophages. Whereas the bacterial gene hly seems responsible for both transcriptional programmes in macrophages, Listeria induces in microglia only the tumor necrosis factor (TNF)‐regulated transcriptional programme. Listeria also represses in microglia the late immune response gathered in two clusters, microbial degradation, and interferon (IFN)‐inducible genes. The bacterial gene actA was required in microglia to induce TNF‐regulated responses and to repress the late response. Isolation of microglial phagosomes revealed a phagosomal environment unable to destroy Listeria. Microglial phagosomes were also defective in several signaling and trafficking components reported as relevant for Listeria innate immune responses. This transcriptional strategy in microglia induced high levels of TNF‐α and monocyte chemotactic protein‐1 and low production of other neurotoxic compounds such as nitric oxide, hydrogen peroxide, and Type I IFNs. These cytokines and toxic microglial products are also released by primary microglia, and this cytokine and chemokine cocktail display a low potential to trigger neuronal apoptosis. This overall bacterial strategy strongly suggests that microglia limit Listeria inflammation pattern exclusively through TNF‐mediated responses to preserve brain integrity. GLIA 2014;62:233–246

Pregnancy Vaccination with Gold Glyco-Nanoparticles Carrying Listeria monocytogenes Peptides Protects against Listeriosis and Brain- and Cutaneous-Associated Morbidities

Listeriosis is a fatal infection for fetuses and newborns with two clinical main morbidities in the neonatal period, meningitis and diffused cutaneous lesions. In this study, we vaccinated pregnant females with two gold glyconanoparticles (GNP) loaded with two peptides, listeriolysin peptide 91–99 (LLO91–99) or glyceraldehyde-3-phosphate dehydrogenase 1–22 peptide (GAPDH1–22). Neonates born to vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice presented clear brain affections and cutaneous diminishment of melanocytes. Therefore, these nanoparticle vaccines are effective measures to offer pregnant mothers at high risk of listeriosis interesting therapies that cross the placenta.