Brucella Control of Dendritic Cell Maturation Is Dependent on the TIR-Containing Protein Btp1

Brucella is an intracellular pathogen able to persist for long periods of time within the host and establish a chronic disease. We show that soon after Brucella inoculation in intestinal loops, dendritic cells from ileal Peyer's patches become infected and constitute a cell target for this pathogen. In vitro, we found that Brucella replicates within dendritic cells and hinders their functional activation. In addition, we identified a new Brucella protein Btp1, which down-modulates maturation of infected dendritic cells by interfering with the TLR2 signaling pathway. These results show that intracellular Brucella is able to control dendritic cell function, which may have important consequences in the development of chronic brucellosis

The Glyceraldehyde-3-Phosphate Dehydrogenase and the Small GTPase Rab 2 Are Crucial for Brucella Replication

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the “Brucella-containing vacuole” (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC ι, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells

Analyse protéomique de la vacuole de Brucella abortus (la glycéraldéhyde-3-phosphate déhydrogénase (GAPDH) et la G Rab 2 sont indispensables pour sa réplication intracellulaire)

Brucella est un pathogène bactérien intracellulaire responsable de la brucellose animale et humaine. Des études in vitro ont démontré que Brucella est capable de contourner la voie d endocytose pour assurer sa survie intracellulaire à la fois dans les cellules phagocytaires et nonphagocytaires. Les brucelles assurent leur réplication par la biogenèse d une vacuole de réplication issue du réticulum endoplasmique (ER) appelée Brucella-containing vacuole (BCV). L identification de protéines spécifiques localisées sur la membrane de la BCV est essentielle pour comprendre comment Brucella est capable de maintenir ces interactions avec le ER et de continuer à se répliquer au sein de ce compartiment. Nous avons caractérisé pour la première fois la composition protéique de sa niche de réplication par des approches protéomiques. Pour cela, nous avons développé une méthode de fractionnement subcellulaire afin d isoler les BCV. L analyse de la composition protéique de la membrane de la BCV par électrophorèse sur gel en 2 dimensions et par spectrométrie de masse a permis l identification de plusieurs protéines eucaryotes et procaryotes. Nous avons obtenu des protéines du ER et des protéines ribosomales qui valident le modèle : la niche de réplication de Brucella est une vacuole qui dérive du ER. De plus, nous avons identifié plusieurs protéines eucaryotes et bactériennes pouvant contribuer à la virulence de Brucella dans les cellules hôtes. Nous avons focalisé notre étude sur 2 protéines eucaryotes : la glyceraldehyde-3-phosphate dehydrogenase (GAPDH) et la petite protéine G Rab 2, qui sont recrutées au niveau de la vacuole de Brucella. Ce complexe a été décrit précédemment pour être localisé sur les vésicules et les tubules de transport (VTC) et pour réguler le trafic des membranes des VTC entre le ER et l appareil de Golgi. La destruction de l appareil de Golgi par des traitements prolongés à la bréfeldine A réduit la réplication de B. abortus. De plus, l inhibition de l expression de GAPDH par l ARN interférence et le blocage de Rab 2 dans sa forme GDP inhibent fortement la réplication de B. abortus. Ces résultats révèlent un rôle essentiel pour GAPDH et Rab 2 dans la virulence de B. abortus à l intérieur des cellules.Brucella is a facultative intracellular pathogenic bacteria which causes severe infections and abortion in animals and a long-lasting febrile disease in humans. Essential to Brucella virulence is its ability to survive and replicate inside host cells. In vitro studies showed that Brucella initially avoids or escapes the endocytic pathway to ensure its intracellular survival both in phagocytic and nonphagocytic cells. Bacteria secure their replication via the biogenesis of an endoplasmic reticulumderived replicative organelle named Brucella-containing vacuole (BCV). Identification of specific proteins on the BCV membrane is essential to understand how Brucella sustain interactions with the endoplasmic reticulum (ER) and keeps replicating within its compartment. We characterize for the first time the protein composition of its replicative niche by proteomic approches. We developed a subcellular fractionation method to isolate BCVs. The analysis of BCV membrane proteins by 2 dimensional gel electrophoresis and mass spectrometry allowed the identification of several eukaryotic and prokaryotic proteins. We obtained ER proteins and ribosomal proteins that validate the model: the replicative niche of Brucella is an ER-derived vacuole. Furthermore, we have identified several interesting host proteins as well as some bacterial proteins that may contribute to Brucella virulence within host cells. We focused our study on two eukaryotic proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2, which are recruited to the vacuolar membrane of Brucella. This complex was previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between ER and Golgi. Disruption of the Golgi apparatus with prolonged brefeldin A treatment reduced B. abortus replication. Furthermore, inhibition of GAPDH expression by small interfering RNA and blockage of Rab 2 GTPase in a GDP-locked form strongly inhibited B. abortus replication. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Virulence factors in brucellosis: implications for aetiopathogenesis and treatment.

International audienceBrucella species are responsible for the global zoonotic disease brucellosis. These intracellular pathogens express a set of factors - including lipopolysaccharides, virulence regulator proteins and phosphatidylcholine - to ensure their full virulence. Some virulence factors are essential for invasion of the host cell, whereas others are crucial to avoid elimination by the host. They allow Brucella spp. to survive and proliferate within its replicative vacuole and enable the bacteria to escape detection by the host immune system. Several strategies have been used to develop animal vaccines against brucellosis, but no adequate vaccine yet exists to cure the disease in humans. This is probably due to the complicated pathophysiology of human Brucella spp. infection, which is different than in animal models. Here we review Brucella spp. virulence factors and how they control bacterial trafficking within the host cell

Identification of living Legionella pneumophila using species-specific metabolic lipopolysaccharide labeling.

International audienceLegionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture-based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O-antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled

Rapid and Specific Enrichment of Culturable Gram Negative Bacteria Using Non-Lethal Copper-Free Click Chemistry Coupled with Magnetic Beads Separation.

Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope

Synthesis of phosphatidylcholine, a typical eukaryotic phospholipid, is necessary for full virulence of the intracellular bacterial parasite Brucella abortus.

Phosphatidylcholine (PC) is a typical eukaryotic phospholipid absent from most prokaryotes. Thus, its presence in some intracellular bacteria is intriguing as it may constitute host mimicry. The role of PC in Brucella abortus was examined by generating mutants in pcs (BApcs) and pmtA (BApmtA), which encode key enzymes of the two bacterial PC biosynthetic routes, the choline and methyl-transferase pathways. In rich medium, BApcs and the double mutant BApcspmtA but not BApmtA displayed reduced growth, increased phosphatidylethanolamine and no PC, showing that Pcs is essential for PC synthesis under these conditions. In minimal medium, the parental strain, BApcs and BApmtA showed reduced but significant amounts of PC suggesting that PmtA may also be functional. Probing with phage Tb, antibiotics, polycations and serum demonstrated that all mutants had altered envelopes. In macrophages, BApcs and BApcspmtA showed reduced ability to evade fusion with lysosomes and establish a replication niche. In mice, BApcs showed attenuation only at early times after infection, BApmtA at later stages and BApcspmtA throughout. The results suggest that Pcs and PmtA have complementary roles in vivo related to nutrient availability and that PC and the membrane properties that depend on this typical eukaryotic phospholipid are essential for Brucella virulence

Schematic representation of metabolic lipopolysaccharide labeling using Kdo-N₃.

<p>Culture of <i>E</i>. <i>coli</i> in the presence of Kdo-N_<b>3</b> results in incorporation of the bioorthogonal <i>azido</i> function with the bacterial LPS. This incorporation can be further visualized by Copper-catalyzed Azide Alkyne Cycloaddition (CuAAC), in the presence of Cu(I) and a terminal alkyne. Alternatively, Strain-Promoted Alkyne Azide Cycloaddition (SPAAC) with a cyclooctyne-type reagent results in copper-free ligation.</p

Kdo-N₃ metabolically labels <i>E</i>. <i>coli</i> LPS.

<p>(A) Metabolically incorporated Kdo-N_<b>3</b> in <i>E</i>. <i>coli</i> was revealed by copper-free click chemistry (sulfo-DBCO-biotin followed by an anti-biotin A488 antibody). (B) Frequency distribution of the bacterial fluorescence values in the presence (green bars) or absence of Kdo-N_<b>3</b> (black bars). Scale bar = 1 μm. More than ten independent experiments have been performed and a representative experiment is depicted.</p

Percentage of culturable bacteria recovery in the supernatant fraction and magnetic streptavidin beads fraction with or without incorporation of Kdo-N₃.

<p>mean in percentage</p><p>Percentage of culturable bacteria recovery in the supernatant fraction and magnetic streptavidin beads fraction with or without incorporation of Kdo-N₃.</p