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

Les maladies à prions. Le « prion » un agent infectieux différent

Les maladies à prions sont des maladies dégénératives du cerveau qui touchent de nombreux mammifères. Elles présentent des caractéristiques épidémiologiques singulières puisqu’elles sont infectieuses, mais aussi génétiques et sporadiques. Ces maladies sont associées à une protéine de l’hôte, la protéine prion qui semble être à la fois l’agent étiologique et une composante physiopathologique essentielle de la maladie. Cette protéine existe sous deux conformations différentes. La forme normale ou cellulaire (PrPc) est riche en hélices-α alors que la forme associée à la maladie ou PrPSc est riche en feuillets-β et résiste à la digestion par la protéinase-K. La conversion de la forme PrPc en une forme résistante à la protéinase-K a été obtenue in vitro, mais son infectiosité n’a pu être démontrée. Pour essayer de rendre compte des phénomènes originaux de ces maladies, plusieurs modèles soit descriptifs, soit moléculaires, ont été proposés

Are prions misfolded molecular chaperones?

AbstractA theory has been developed that could explain prion infection. Prions could be molecular chaperones that are required for their own assembly. The theory has been deduced from an analysis of protein folding and consequences explored by computer simulations. Thermo-kinetic analysis of protein folding shows that a misfolded chaperone gives rise to new misfolded chaperones. Consequently such a protein could behave as a new kind of informative molecule and replicate misfolding according to a process similar to infection. A quantitative model has been derived from this hypothesis that displays the characteristics of prion infections. This hypothesis satisfactorily explains the three manifestations infection, familial and sporadic — that are the characteristics features of all prion diseases

Identification et caractérisation des ClpATPases ClpA & ClpB de la bactérie pathogène Brucella Suis

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Etude fonctionnelle de la protéine prion cellulaire dans deux populations de cellules immunitaires

Les Encéphalopathies Spongiformes Subaiguës Transmissibles (ESST) sont des pathologies neurodégénératives qui présentent des déterminismes multiples. On distingue ainsi, les ESST familiales, sporadiques et infectieuses. Quelque soit leur forme, ces maladies sont généralement associées à une protéine appelée protéine du prion ou PrP. Notamment dans les ESST infectieuses, un homologue conformationnel de cette protéine est suspecté d être le constituant principal de l agent infectieux. Les deux formes de PrP sont identifiées sous les termes de cellulaire (PrPC), pour la protéine native et de scrapie (PrPSc), pour son homologue pathologique. Beaucoup d incertitudes planent sur la physiopathologie associée aux ESST, notamment parce que la fonction physiologique de la PrPC n est pas connue précisément. La protéine prion cellulaire est une glycoprotéine, ancrée via un groupement GlycosylPhosphatidylInositol, dans le feuillet externe de la membrane. La fonction physiologique de la PrP a été abondamment étudiée et la protéine a été impliquée dans de nombreux mécanismes cellulaires comme la transduction de signaux, la modulation de l apoptose, l adhésion cellulaire ou la reconnaissance des bactéries du genre Brucella. Cependant, beaucoup de ces études sont controversées et aucune ne fait l unanimité. Nous nous sommes intéressés, à notre tour, à la fonction physiologique de la PrPC dans deux modèles de cellules immunitaires : les lymphocytes Tg9 2 humains, sur lesquels nous avons observé une expression membranaire de la protéine très abondante et les macrophages murins, qui ont été impliqués dans la reconnaissance et la survie intracellulaire des Brucella. Nous avons étudié l éventuelle participation de la PrPC dans les divers mécanismes physiologiques associés à ces lymphocytes, d une part et à l infection des macrophages par Brucella, d autre part. Or, les résultats obtenus n ont pas permis de dégager une fonction physiologique évidente de la PrPC dans ces cellulesPrion diseases are neurodegeneratives pathologies of multiples determinisms. Inherited, sporadic or infectious forms are all associated with the expression of one protein, called prion protein or PrP. Infectious diseases are suspected to be caused by an abnormal conformational isoform of the cellular prion protein (PrPC) named scrapie or PrPSc . Physiopathologic events associated with prion diseases are not completely understood, in part because physiological function of PrPC is not precisely known. PrPC is a glycoprotein located in the extracellular side of the membrane with GPI anchorage. Due to its location, PrPC has been implicated in many cellular functions as signalling, apoptosis modulation, cellular adhesion or receptor for the bacteria Brucella. However, many of these functions are not clear or are controversial. We try to determine the physiological role of cellular prion protein by studying different cellular mechanisms of two populations of immune cells : Tg9 2 lymphocytes in which important expression of PrP has been found and murine macrophages where PrP has been implicated in the entry and intracellular proliferation of Brucella. But, in both cases, we were unable to demonstrate a functional implication of the cellular prion proteinMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Targeting of the Virulence Factor Acetohydroxyacid Synthase by Sulfonylureas Results in Inhibition of Intramacrophagic Multiplication of Brucella suis

The acetohydroxyacid synthase (AHAS) of Brucella suis can be effectively targeted by the sulfonylureas chlorimuron ethyl and metsulfuron methyl. Growth in minimal medium was inhibited, and multiplication in human macrophages was totally abolished with 100 μM of sulfonylureas. Metsulfuron methyl-resistant mutants showed reduced viability in macrophages and reduced AHAS activity

Live Brucella spp. fail to induce tumor necrosis factor alpha excretion upon infection of U937-derived phagocytes.

Tumor necrosis factor alpha (TNF-alpha) plays a central role in activation of first-line defenses of a host against foreign organisms. To determine whether Brucella infection modulated TNF-alpha production, we measured the biological activity of this cytokine in supernatants of U937 cell-derived macrophages and of fresh human monocytes infected with Brucella spp. Neither the smooth nor rough Brucella strains used induced any measurable TNF-alpha excretion upon infection. On the contrary, as reported before for other gram-negative bacteria, phagocytosis of nonpathogenic Escherichia coli was followed by a rapid and transient induction of TNF-alpha release, suggesting an involvement of this cytokine in some autocrine process. As expected, the Brucella strains tested survived and/or multiplied within U937-derived macrophages, whereas E. coli was rapidly eliminated after phagocytosis. Immunoglobulin G opsonization of E. coli strains enhanced their intracellular killing and strongly potentiated TNF-alpha secretion. Immunoglobulin G opsonization of Brucella strains, in contrast, did not lead to TNF-alpha production, although their rate of intracellular multiplication was reduced. Killed brucellae, however, promoted a significant excretion of TNF-alpha from U937-derived macrophages into cell culture supernatants. We finally demonstrated that pretreatment of U937-derived macrophages with exogenous TNF-alpha significantly inhibited intracellular multiplication of Brucella spp. These results and experiments performed on fresh human monocytes or with isolated lipopolysaccharide (LPS) showed that (i) differences in TNF-alpha production observed during macrophage infection by Brucella spp. and E. coli were not due to differences in LPS structure but resulted from active inhibition of TNF-alpha production by a specific process linked to Brucella spp. and (ii) the capacity of Brucella spp. to use pathways avoiding TNF-alpha production during infection may be considered a major attribute of virulence