Date : 03/03/2011

Laboratory
Equipe Avenir - Pathogenèse infectieuse des vaisseaux
U970 INSERM
56 Rue Leblanc
75015 Paris
Website : http://parcc.inserm.fr
Main discipline : Cell Biology
Lab director : Alain Tedgui

PhD Supervisor
Guillaume Dumenil
email : This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
phone : +33 1 53 98 80 49

Subjects
1.: Meningitis
2.: Cerebral microcirculation
3.: Neisseria meningitidis

Tools and methodologies
1.: Cellular microbiology
2.: Microfluidics
3.: Live imaging

Summary of lab's interests

infectious diseases remain a major public health problem in particular because of the emergence of antibiotic resistant pathogens. Our ability to design new treatments depends on increased understanding of the mechanisms of disease. Our group focuses on a subset of infectious diseases that take place in the vasculature. Infections taking place in the bloodstream are usually extremely severe and often life threatening because they lead to septic choc and meningitis for instance. We study different such pathogens with a particular focus on the bacterium Neisseria meningitidis, also called meningococcus, responsible for septicemia and meningitis. Studying these particular infections requires specific approaches mimicking the conditions found in the human vasculature. We use a combination of techniques originating from cell biology, microbiology, vascular biology chemistry and physics to reproduce experimentally the interaction between these pathogens and the vascular wall. This strategy allows us to collect information on the properties of these pathogens and their target during the infection process that will guide the design of innovative therapeutic approaches.

Summary of project

The bacterium Neisseria meningitidis is responsible for meningitis in humans. During pathogenesis N. meningitidis is able to enter blood vessels, access the global circulation and invade the brain by exiting cerebral capillaries. How this bacterium exits the lumen of capillaries to access the brain is unknown. To address this point efficiently it is necessary to reconstitute the key properties of the blood-brain barrier. One of these characteristics is a specific geometry. Vessels targeted by N. meningitidis infection characteristically display sizes in the order of 10 µm. Bacteria such as N. meningitidis display sizes around 1 µm but the bacteria live as aggregates that can potentially reach sizes superior to the capillary diameter. As a consequence, infection can cause occlusion of the infected vessel and subsequently affect its physiology. To mimic the environment of vascular infection it is therefore important to find ways to reproduce the scale of small vessels, which represents an experimental challenge. For this purpose we will take advantage of the recent advances in the field of microfluidics, which allows the microfabrication of flow chambers of sizes compatible with capillaries. In collaboration with the group headed by Olivier Theodoly we are developing microfabricated devices to achieve this goal. At the current stage, devices are made of a central channel of 150 μm in size decorated with numerous side channels of very small width (around 2 μm). This series of side channels will create an apparent "porosity" of the walls of the central channel as the eventual flow across these porous walls is drained by lateral collector channels. Endothelial cells will be cultivated in the central 150 μm channel and bacteria will be injected into the central channel at different flow rates. Bacteria exiting the "vessel" lumen will be collected in the lateral channels. The exit process will also be analyzed by live fluorescence microscopy (spinning disc confocal). This model will be used in particular to evaluate the role of PtpB, a recently described bacterial enzyme in the process of blood-brain barrier crossing by comparing the behavior of a mutant with the wild type. The involvement of other bacterial and cellular components can be tested in this system to obtain a global picture of the process.

Interdisciplinarity of the project

1) Cellular microbiology The basis of this project is the interaction of bacteria with cerebral microvessels. Microbiology, cellular biology and vascular biology are thus at the core of this project. 2) Physics The experimental approach of this project depends on a close collaboration with the team headed by Olivier Theodoly in Marseille-Luminy specialized in the field of biophysics and more specifically microfluidics. Microfluidics chambers mimicking cerebral capillaries will be designed and made in this laboratory with the participation of the PhD student. 3) Live imaging Live imaging techniques will also be an important element of this project to visualize the infection process (spinning disc confocal micrcoscopy). 4) Analytical chemistry The function of PptB, a recently described bacterial enzyme will be at the center of this project. The activity of this enzyme is evaluated by advanced mass spectrometry techniques in close collaboration with the group headed by Julia Chamot-Rooke at the Ecole Polytechnique (top-down whole protein approach, FT-ICR, Q-TOF...).