A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly

P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. P-bodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function

Bacteria Modulate the CD8+ T Cell Epitope Repertoire of Host Cytosol-Exposed Proteins to Manipulate the Host Immune Response

The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested

A-Kinase Anchoring in Dendritic Cells Is Required for Antigen Presentation

BACKGROUND: Dendritic cells (DC) are the most potent antigen presenting cells (APC) of the immune system. Prostaglandin E(2), cyclic AMP, and protein kinase A (PKA) have all been shown to regulate DC maturation and activity. In other cells, the ability of these molecules to convey their signals has been shown to be dependent on A-kinase anchoring proteins (AKAPs). Here we present evidence for the existence and functional importance of AKAPs in human DC. METHODOLOGY/PRINCIPAL FINDINGS: Using immunofluorescence and/or western analyses we identify AKAP79, AKAP149, AKAP95, AKAP LBC and Ezrin. We also demonstrate by western analysis that expression of AKAP79, AKAP149 and RII are upregulated with DC differentiation and maturation. We establish the functional importance of PKA anchoring in multiple aspects of DC biology using the anchoring inhibitor peptides Ht31 and AKAP-IS. Incubation of protein or peptide antigen loaded DC with Ht31 or AKAP-IS results in a 30-50% decrease in antigen presentation as measured by IFN-gamma production from antigen specific CD4(+) T cells. Incubation of LPS treated DC with Ht31 results in 80% inhibition of TNF-alpha and IL-10 production. Ht31 slightly decreases the expression of CD18 and CD11a and CD11b, slightly increases the basal expression of CD83, dramatically decreases the LPS stimulated expression of CD40, CD80 and CD83, and significantly increases the expression of the chemokine receptor CCR7. CONCLUSIONS: These experiments represent the first evidence for the functional importance of PKA anchoring in multiple aspects of DC biology

Automated Negotiation Mechanism and Strategy for Compensational Vehicular Platooning

Our research is developing flexible strategies for forming and routing future platoons of automated urban logistics vehicles. We propose the notion of compensational platooning using automated negotiation between agents representing vehicles. After the vehicles reach the end of a common route, an agent can propose part of its route along with a monetary value to platoon partners for further together-travel. If negotiation is successful, a new platoon is formed and follows the proposed route. If the compensation is too small or the route proposed oversteps the agent's limitations, the offer is rejected and the vehicles continue their travel separately. A contribution of this paper is a negotiation strategy that proposes compensation based on beliefs of what the opponent's payment threshold would be. In doing so, the bid with the highest acceptance likelihood is calculated, keeping negotiations short and effective. Our model is tested on a synthetic network and a real urban example. We show that by using negotiation, vehicles can identify mutually beneficial new routes that a centralised/distributed approach would not find, with utility improvements of up to 8%

A Salmonella virulence protein that inhibits cellular trafficking.

Salmonella enterica requires a type III secretion system, designated Spi/Ssa, to survive and proliferate within macrophages. The Spi/Ssa system is encoded within the SPI-2 pathogenicity island and appears to function intracellularly. Here, we establish that the SPI-2-encoded SpiC protein is exported by the Spi/Ssa type III secretion system into the host cell cytosol where it interferes with intracellular trafficking. In J774 macrophages, wild-type Salmonella inhibited fusion of Salmonella-containing phagosomes with lysosomes and endosomes, and interfered with trafficking of vesicles devoid of the microorganism. These inhibitory activities required living Salmonella and a functional spiC gene. Purified SpiC protein inhibited endosome-endosome fusion in vitro. A Sindbis virus expressing the SpiC protein interfered with normal trafficking of the transferrin receptor in vivo. A spiC mutant was attenuated for virulence, suggesting that the ability to interfere with intracellular trafficking is essential for Salmonella pathogenesis

Automated Negotiation Mechanism and Strategy for Compensational Vehicular Platooning

Our research is developing flexible strategies for forming and routing future platoons of automated urban logistics vehicles. We propose the notion of compensational platooning using automated negotiation between agents representing vehicles. After the vehicles reach the end of a common route, an agent can propose part of its route along with a monetary value to platoon partners for further together-travel. If negotiation is successful, a new platoon is formed and follows the proposed route. If the compensation is too small or the route proposed oversteps the agent's limitations, the offer is rejected and the vehicles continue their travel separately. A contribution of this paper is a negotiation strategy that proposes compensation based on beliefs of what the opponent's payment threshold would be. In doing so, the bid with the highest acceptance likelihood is calculated, keeping negotiations short and effective. Our model is tested on a synthetic network and a real urban example. We show that by using negotiation, vehicles can identify mutually beneficial new routes that a centralised/distributed approach would not find, with utility improvements of up to 8%

Brucella suis-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments

Salmonella enterica Serovar Gallinarum Requires the Salmonella Pathogenicity Island 2 Type III Secretion System but Not the Salmonella Pathogenicity Island 1 Type III Secretion System for Virulence in Chickens

Salmonella enterica serovar Gallinarum is a host-specific serotype that causes the severe systemic disease fowl typhoid in domestic poultry and a narrow range of other avian species but rarely causes disease in mammalian hosts. Specificity of the disease is primarily at the level of the reticuloendothelial system, but few virulence factors have been described other than the requirement for an 85-kb virulence plasmid. In this work, by making functional mutations in the type III secretion systems (TTSS) encoded by Salmonella pathogenicity island 1 (SPI-1) and SPI-2, we investigated the role of these pathogenicity islands in interactions between Salmonella serovar Gallinarum and avian cells in vitro and the role of these pathogenicity islands in virulence in chickens. The SPI-1 mutant showed decreased invasiveness into avian cells in vitro but was unaffected in its ability to persist within chicken macrophages. In contrast the SPI-2 mutant was fully invasive in nonphagocytic cells but failed to persist in macrophages. In chicken infections the SPI-2 mutant was attenuated while the SPI-1 mutant showed full virulence. In oral infections the SPI-2 mutant was not observed in the spleen or liver, and following intravenous inoculation it was cleared rapidly from these sites. SPI-2 function is required by Salmonella serovar Gallinarum for virulence, primarily through promoting survival within macrophages allowing multiplication within the reticuloendothelial system, but this does not preclude the involvement of SPI-2 in uptake from the gut to the spleen and liver. SPI-1 appears to have little effect on virulence and survival of Salmonella serovar Gallinarum in the host