Caractérisation du second système de sécrétion de type II chez la bactérie phytopathogène Erwinia chrysanthemi

Chez les bactéries à Gram négatif, toutes les protéines destinées à la membrane externe sont synthétisées avec une séquence signal qui est clivée lors de leur acheminement. Ce clivage seffectue lors du passage de la membrane interne, par LepB pour les protéines intégrales de la membrane externe ou par LspA pour les lipoprotéines. Les séquençage du génome de Dickeya dadantii a permis de mettre en évidence une seconde machinerie de sécrétion de type II nommée Stt (pour Second Type Two). En aval de ce système se trouve un gène dénommé pnlH dont le produit est homologue à des pectines lyases. Létude de cette protéine a montré quelle est un substrat de la machinerie Stt qui permet son passage de la face interne de la membrane externe à sa face externe. En labsence de Stt ou chez Escherichia coli, PnlH est localisée à la face interne de la membrane externe. Cet ancrage est dû à lextrémité N-terminale de la protéine qui nest pas clivée lors du passage de la membrane interne et contient toute linformation pour ladressage de la protéine. En effet, la fusion des 41 premiers acides aminés de PnlH à des protéines de différents compartiments cellulaires provoque ladressage de celle-ci à la membrane externe. Lanalyse plus approfondie de cette partie N-terminale montre certaines caractéristiques dune séquence signal Tat-dépendante, permettant le passage de la membrane interne par le système Tat. Lanalyse de mutants de la séquence signal ou de la machinerie Tat a confirmé que celle-ci est nécessaire pour le transfert de PnlH à travers la membrane interne. Ainsi, lanalyse de ladressage de PnlH à la membrane externe a permis de mettre en évidence une nouvelle voie dacheminement de protéines à la membrane externe des bactéries. Ce nouveau mécanisme de ciblage de protéines à la membrane externe est probablement répandu puisque PnlH est aussi localisée à la membrane externe quand elle est exprimée dans le E. coli. PnlH nétant pas détectée comme substrat de la machinerie Tat par les programmes de prédiction, cela suggère quil reste à découvrir dautres protéines de la membrane externe Tat-dépendante encore non identifiées

Ctenocephalides felis an in vitro potential vector for five Bartonella species

International audienceThe blood-sucking arthropod Ctenocephalides fells has been confirmed as a vector for Bartonella henselae and is a suspected vector for Bartonella clarridgeiae, Bartonella quintana and Bartonella koehlerae in Bartonella transmission to mammals. To understand the absence of other Bartonella species in the cat flea, we have developed an artificial flea-feeding method with blood infected successively with five different Bartonella species. The results demonstrated the ability of these five Bartonella species to persist in C. felis suggesting an ability of fleas to be a potential vector for several Bartonella species. In addition, we demonstrated a regurgitation of Bartonella DNA in uninfected blood used to feed C. felis thus suggesting a potential horizontal transmission of Bartonella through C. felis saliva. On the contrary, no vertical transmission was detected in these artificial conditions. (c) 2012 Elsevier Ltd. All rights reserved

Allosteric inhibition of the guanine nucleotide exchange factor DOCK5 by a small molecule

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Heme binding proteins of <em>Bartonella henselae</em> are required when undergoing oxidative stress during cell and flea invasion

International audienceBartonella are hemotropic bacteria responsible for emerging zoonoses. These heme auxotroph alphaproteobacteria must import heme for their growth, since they cannot synthesize it. To import exogenous heme, Bartonella genomes encode for a complete heme uptake system enabling transportation of this compound into the cytoplasm and degrading it to release iron. In addition, these bacteria encode for four or five outer membrane heme binding proteins (Hbps). The structural genes of these highly homologous proteins are expressed differently depending on oxygen, temperature and heme concentrations. These proteins were hypothesized as being involved in various cellular processes according to their ability to bind heme and their regulation profile. In this report, we investigated the roles of the four Hbps of Bartonella henselae, responsible for cat scratch disease. We show that Hbps can bind heme in vitro. They are able to enhance the efficiency of heme uptake when co-expressed with a heme transporter in Escherichia coli. Using B. henselae Hbp knockdown mutants, we show that these proteins are involved in defense against the oxidative stress, colonization of human endothelial cell and survival in the flea

Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis

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Pharmacological inhibition of Dock5 prevents osteolysis by affecting osteoclast podosome organization while preserving bone formation

International audienceOsteoporosis is caused by excessive activity of bone-degrading osteoclasts over bone-forming osteoblast. Standard antiosteolytic treatments inhibit bone resorption by inducing osteoclast loss, with the adverse effect of hindering also bone formation. Formation of the osteoclast sealing zone requires Dock5, a guanine nucleotide exchange factor for the small GTPase Rac, and C21, a chemical inhibitor of Dock5, decreases bone resorption by cultured osteoclasts. Here we show that C21 directly inhibits the exchange activity of Dock5 and disrupts osteoclast podosome organization. Remarkably, C21 administration protects mice against bone degradation in models recapitulating major osteolytic diseases: menopause, rheumatoid arthritis and bone metastasis. Furthermore, C21 administration does not affect bone formation and is not toxic. Our results validate the pharmacological inhibition of Dock5 as a novel therapeutic route for fighting osteolytic diseases while preserving bone formation

Hbp knockdown decreases the ability of <i>B. henselae</i> to undergo exposure to H₂O₂.

<p><i>B. henselae</i> pNS2Trc and <i>B. henselae</i> pNS2Trc::<i>hbps</i>_AS were challenged with 10 mM H₂O₂ as described in Materials and methods. Experiments were performed in triplicate; survival rates were expressed as described in Materials and methods. (*P<0.05, **P<0.01 compared to <i>B. henselae</i> pNS2Trc).</p

Detection of <i>B. henselae</i> DNA from flea feces samples using PCR.

<p>About 500 fleas were first feed with blood containing 500 µl bacteria (2×10⁸/ml) for 2 days and then fed uninfected blood for 8 days. Flea feces were collected every day. DNA was extracted from flea feces and PCR was performed as described in Materials and methods.</p

Effect of <i>hbps</i> knockdown on growth of <i>B. henselae</i> on blood plates.

<p>For the growth test on CBA plates, strains <i>B. henselae</i> (pNS2Trc), <i>B. henselae</i> (pNS2Trc<i>::hbpA _AS</i>), <i>B. henselae</i> (pNS2Trc<i>::hbpB _AS</i>), <i>B. henselae</i> (pNS2Trc<i>::hbpC _AS</i>) and <i>B. henselae</i> (pNS2Trc<i>::hbpD_AS</i>) were collected after 5 days of growth on CBA plates and suspended in PBS buffer to obtain about 10³ CFU ml⁻¹. Two-hundred microliters of cell suspension were plated on the CBA plate. Colony sizes were measured after 6 and 10 days of growth at 35°C in the presence of 5% CO₂. Data are the mean diameter (mm) ± SD of 10 colonies from one representative experiment. Standard deviations were calculated using Statview software. All experiments were repeated three times. NM: not measurable.</p

Effect of Hbp knockdown on endothelial cell invasion.

<p>Invasion of endothelial cells by <i>B. henselae</i> pNS2Trc and <i>B. henselae</i> pNS2Trc<i>::hbps_AS.</i> Cells were mixed with bacteria at 0.1 m.o.i. After 24 h, infected cell were treated with gentamicin to kill extracellular bacteria and lysates were plated on the CBA blood plate to determine the number of intracellular bacteria. Invasion was calculated using the equation provided in Materials and methods. (***P<0.005 compared to <i>B. henselae</i> pNS2Trc).</p