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Manipulation of membrane traffic by bacterial pathogens – Bacterial-Tactics

Bacterial tactics

Cellular traffic is frequently hijacked by intracellular pathogens to invade the cell and replicate. Small GTPases are major regulators of cellular traffic, and are often the target of bacterial toxins. We study the structural and biochemical determinants of bacterial toxins that hijack small GTPases.

structural and biochemical determinants of bacterial toxins that hijack small GTPases

Characterize the structural and biochemical determinants of toxins from bacterial pathogens that hijack small GTPases pathways; study their interactions at the surface of membarnes

Recombinant proteins
X-Ray crystallography
SAXS
Liposomes
Fluorescence spectroscopies
Lipid cubic phases

We study proteins from Legionella pneumophila et de Coxiella burnetti. We have characterized structural features, interactions with small GTPases and membranes, and developped an innovative strategy for the crystallographic analysis of peripheral membrane proteins

We have discovered outstanding properties of toxins tthat are explained by their structural organization

Plusieurs manuscrits décrivant ces résultats sont en cours d'écriture

To invade their host and avoid from being destroyed, intracellular bacterial pathogens inject numerous proteins (collectively called effectors) which exert biochemical functions to take command of host cell pathways. Membrane traffic is among the primary pathways manipulated by these effectors, allowing pathogens to escape from the phago-lysosomal pathway or to convert phagosomes into specialized compartments where they hide and replicate. Understanding the molecular tactics that pathogens use to subvert trafficking machineries is an important issue to elucidating how they survive in the infected cell, which can inspire novel therapeutic strategies to combat infections. The mechanisms of effectors from Legionella pneumophila (Lp) and from the phylogenetically related pathogens Coxiella burnetti (Cb) and Rickettsia prowazekii (Rp), which manipulate membrane traffic are being investigated in this project, using an interdisciplinary approach that combines reconstitution of effectors and their cellular targets on artificial membranes, structural biology by classical and in meso crystallization and cellular microbiology.

L. pneumophila is responsible for the Legionnaire’s disease, an acute pneumonia transmitted via ill-maintained water systems. It infects lung macrophages, where it evades destruction by camouflaging in a specialized vacuole that originates from the phagosome and rapidly diverts from the degradative lysosomal pathway by incorporating membranes and proteins from the endoplasmic reticulum. To create this vacuole and persist in it, Lp uses a type IV secretion system to deliver over 280 effectors, a number of which target host cell traffic. A hallmark of Lp is the subversion of Arf and Rab small GTPases (which are chief organizers of membrane traffic) by effectors that mimic host regulators or carry out reversible post-translational modifications. The mechanisms of Lp effectors that manipulate the localization and activity of trafficking GTPases by post-translational modifications is being investigated in this project.

C. burnetti is an extremely resistant organism which causes Q-fever, a worldwide disease with acute and chronic stages that is transmitted to humans via aerosols from contaminated soils or livestock. Cb also hijacks trafficking pathways of the cell to establish a specialized membrane-bound organelle, but uses a very different strategy. The Cb-containing vacuole (CCV) derives from fusion of the phagosome with lysosomal vesicles, thus providing the pathogen with an acidic PH environment that enables its intracellular replication. One of the most striking features of the mature CCV is its ability to fuse promiscuously with other lysosome-derived vacuoles in the host cell, which creates a spacious vacuole that contains all of the intracellular bacteria. The structure and function of a novel Cb effector that manipulates autophagy processes to promote the fusogenic properties of the CCV are being investigated in this project.

An important function of bacterial effectors that manipulate trafficking pathways is to relocate host GTPases to illegitimate membranes and/or to exclude these GTPases from cellular membranes. The underlying mechanisms rely on their recognition of specific membranes, where they acquire their active structure. Visualizing these interactions and conformational changes at high resolution in the crystal is extremely difficult. The use of in meso phase crystallization to mimic the membrane interface in the crystal is investigated in this project, focusing as a model system on a membrane-regulated effector that activates host GTPases in Legionella and in Rickettsia, the causative agent of epidemic typhus.

This ensemble of studies should deliver a high resolution, integrated understanding of the molecular mechanism of effectors that intracellular pathogens use to manipulate membrane traffic and autophagy, as well as new structural methods to study their interactions with membranes.

Project coordinator

Madame Jacqueline CHERFILS (Laboratoire de Biologie et Pharmacologie Appliquée)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS - LBPA Laboratoire de Biologie et Pharmacologie Appliquée
IBS - UJF Institut de Biologie Structurale - Université Joseph Fourier
Yale University Department of Microbiology
CNRS - LBPA Laboratoire de Biologie et Pharmacologie Appliquée

Help of the ANR 482,971 euros
Beginning and duration of the scientific project: September 2014 - 36 Months

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