Brucella Evades Macrophage Killing via VirB-dependent Sustained Interactions with the Endoplasmic Reticulum

The intracellular pathogen Brucella is the causative agent of brucellosis, a worldwide zoonosis that affects mammals, including humans. Essential to Brucella virulence is its ability to survive and replicate inside host macrophages, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here we show in a model of Brucella abortus infection of murine bone marrow–derived macrophages that a fraction of the bacteria that survive an initial macrophage killing proceed to replicate in a compartment segregated from the endocytic pathway. The maturation of the Brucella-containing vacuole involves sustained interactions and fusion with the endoplasmic reticulum (ER), which creates a replicative compartment with ER-like properties. The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport. A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes. Thus, we demonstrate that live intracellular Brucella evade macrophage killing through VirB-dependent sustained interactions with the ER. Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle

Initiation of V(D)J Recombination by Dβ-Associated Recombination Signal Sequences: A Critical Control Point in TCRβ Gene Assembly

T cell receptor (TCR) β gene assembly by V(D)J recombination proceeds via successive Dβ-to-Jβ and Vβ-to-DJβ rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vβ-to-Jβ rearrangements. However the B12/23 restriction does not explain the order of TCRβ assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRβ locus and found that nicks were only detectable at Dβ-associated RSSs. This pattern implies that Dβ 12RSS and, unexpectedly, Dβ 23RSS initiate V(D)J recombination and capture their respective Vβ or Jβ RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dβ1 23RSS impedes cleavage at the adjacent Dβ1 12RSS and consequently Vβ-to-Dβ1 rearrangement first requires the Dβ1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a ‘Dβ1 23RSS-mediated’ restriction operating beyond chromatin accessibility, which directs Dβ1 ordered rearrangements

RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement

V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus

Rôle des séquences signal de recombinaison dans le contrôle des réarrangements au locus TCRb

Les gènes codant pour les récepteurs antigéniques (TCR et Ig) sont assemblés par un processus de réarrangement somatique dénommé Recombinaison V(D)J. Ce mécanisme est ciblé au niveau de l ADN par les séquences signal de recombinaison (RSS). Ce processus est en grande partie régulé par la structure de la chromatine. Cependant, d autres mécanismes existent, qui opèrent au-delà de l accessibilité, pour réguler plus finement la recombinaison. Un exemple est la restriction B12/23 qui inhibe, malgré la compatibilité 12/23, les réarrangements directs Vb-Jb, et par conséquent assure l incorporation du segment intermédiaire Db. L objectif de ma thèse était de comprendre l impact qu ont les RSS sur l efficacité et la spécificité des recombinaisons V(D)J. J ai focalisé mon étude sur les RSS du TCRb et pour m affranchir des contraintes chromatiniennes, j ai développé un système d étude des clivages in vitro. J ai défini la base mécanistique de la restriction B12/23, mais j ai aussi mis en évidence le rôle majeur de la 23-RSS 3 Db1. En effet, cette RSS dirige l ordre des réarrangements au locus TCRb. Finalement, mes résultats indiquent qu un facteur, exprimé dans les stades immatures de la différenciation T serait impliqué dans la spécificité cellulaire des réarrangements et l exclusion allélique au TCRb. En conclusion, ma thèse contribue à la compréhension des différents mécanismes qui régulent, au-delà de l accessibilité, la recombinaison V(D)J.V(D)J recombination, the process by which antigen receptor is assembled, is targeted by the recombination signal sequences (RSS) that flank each V, D and J coding segment. This process is largely regulated by chromatin structure. However some mechanisms that operate beyond accessibility exist to fine-tune V(D)J recombination regulation. The first example is the B12/23 restriction that impedes, despite 12/23 compatibility, the direct rearrangement between Vb and Jb gene segments; therefore the B12/23 insures the Db segment incorporation. The aim of my thesis was to understand the impact of RSS on VDJ recombination efficiency and specificity. I focused my study on TCRb RSS and to get free from chromatin, I developed an in vitro cleavage assay system. I defined the mechanistic basis of the B12/23 restriction, but also I uncovered a major role for the 23-RSS Db1. Indeed, this RSS dictates a rearrangement order at the TCRb locus. Finally my results point out that a factor expressed in immature DN T-cells, stimulate RAG cleavage activity. This factor is not yet characterized; it might be involved in the cellular specificity of the rearrangement and allelic exclusion at the TCRb locus. To conclude, my thesis contributes to the understanding of these various mechanisms that regulates, beyond accessibility, the V(D)J recombination.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Contrôle de l’activité lymphocytaire par les granules de stress

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Regulatory Mechanisms of Inhibitory Immune Checkpoint Receptors Expression

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Simultaneous in vitro characterisation of DNA deaminase function and associated DNA repair pathways.

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes

Stress Granules in the Post-transcriptional Regulation of Immune Cells

International audienceImmune cell activation triggers transcriptional and translational programs eliciting cellular processes, such as differentiation or proliferation, essential for an efficient immune response. These dynamic processes require an intricate orchestration of regulatory mechanisms to control the precise spatiotemporal expression of proteins. Post-transcriptional regulation ensures the control of messenger RNA metabolism and appropriate translation. Among these post-transcriptional regulatory mechanisms, stress granules participate in the control of protein synthesis. Stress granules are ribonucleoprotein complexes that form upon stress, typically under control of the integrated stress response. Such structures assemble upon stimulation of immune cells where they control selective translational programs ensuring the establishment of accurate effector functions. In this review, we summarize the current knowledge about post-transcriptional regulation in immune cells and highlight the role of stress sensors and stress granules in such regulation