Cells communicate with their environment through membrane receptors which recognize molecules and particles in the external milieu. These interactions take place at the plasma membrane and may induce intracellular signals or even allow the entry of pathogens into the cell. Our research is focused on the entry and intracellular traffic in eukaryotic cells of membrane receptors and intracellular bacteria, the Chlamydia. The receptors studied are expressed on lymphocytes and are essential for the immune response : the T lymphocyte antigen receptor and the receptors of a cytokine, the interleukin 2. Depending on the strain, the chlamydiae are the causative agents of sexually transmitted diseases, pulmonary infections and eye infections, and they may also be involved in atherosclerosis. We are currently characterizing their entry into and development within host cells.
1. RECEPTOR ENDOCYTOSIS AND INTRACELLULAR TRAFFIC(A. Dautry-Varsat, A. Alcover)
Membrane receptors recognize their extracellular ligand and the ligand-receptor complexes are then endocytosed. Receptors and ligands are then sorted in intracellular membrane compartments. Endocytosis leads to changes in the expression, function and localisation of membrane components.
Receptor mediated endocytosis allows cells to communicate with their environment via membrane receptors which bind macromolecules in the extracellular milieu. It is an essential process for cell homeostasy since it controls many functions including nutriment uptake, growth factor and hormone responses, antigen presentation and the entry of some pathogens.
Receptor-mediated endocytosis through clathrin-coated pits and vesicles has been by far the most thoroughly investigated. Receptor-ligand complexes concentrate in coated pits which invaginate to form coated vesicles that bud from the plasma membrane and rapidly loose their coat. These vesicles then fuse with intracellular compartments named endosomes.
We have developped new tools to specifically and efficiently block clathrin-mediated endocytosis : dominant negative mutants of the protein Eps15. This has allowed to identify interleukin 2 (IL2) as the first physiological ligand endocytosed by a new clathrin-independent mechanism. IL2 receptor endocytosis is rapid and efficient. IL2 receptors could not be detected in clathrin-coated structures on the plasma membrane, unlike transferrin receptors which are markers of the clathrin-dependent endocytosis pathway. Instead, IL2 receptors were found constituvely associated with membrane microdomains enriched in cholesterol and sphingolipids, named "rafts". The receptors are still in rafts when they reach endosomes. Finally, this new endocytosis pathway is specifically regulated by Rho family GTPases, while the GTPase dynamin is involved both in clathrin-dependent and independent endocytosis.
Sorting of intracellular receptors : a role for ubiquitin
After internalization, IL2 receptors reach endosomes where they are sorted and targeted to lysosomes and degraded. Interestingly, one of the receptor's chains is mono-ubiquitinated ; if its ubiquitination is prevented, its degradation is also inhibited while its internalization is not affected. Furthermore, the IL2 receptor contains a small amino-acid sequence, responsible for its sorting to lysosomes which is sufficient, when added to a chimeric membrane protein, to target it to lysosomes, once it is internalized.
In conclusion, the ubiquitination machinery, in addition to its well established role in allowing proteasome degradation of nuclear and cytosolic proteins, is also involved in intracellular sorting of receptors thus controlling their surface expression.
T lymphocyte response to bacterial superantigens
Some bacterial toxins responsible for food poisoning, induce a strong T cell activation and are responsible for pathologies. These toxins, called superantigens, can induce a strong immune response even when present in small amounts. This is due to their recognition by T lymphocytes. We have shown that this strong T lymphocyte response may result from a cooperative activation of T cell receptors on the plasma membrane. Moreover, toxin superantigens induce changes in the T cell morphology and polarisation, due to a profound reorganisation of membrane components and of the cytoskeleton (Figure 1). We have observed that proteins that establish links between the plasma membrane and the cytoskeleton are essential for T lymphocyte activation.
2. Invasion BY INTRACELLULAR BACTERIA, CHLAMYDIA(A. Dautry-Varsat, I. Jutras, A. Subtil, B. Wyplosz)
Chlamydiae are bacteria that proliferate only within eukaryotic host cells. The three species pathogenic to humans, Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae, cause a number of diseases, including trachoma, pelvic inflammatory disease, pneumonia and trachoma.
Primary infections are often minor or asymptomatic; the sequelae, blindness, sterility or ectopic pregnancy appear long after infection. Throughout their cycle in the host cell, chlamydiae remain in a membrane-bound compartment called an inclusion (Figure 2). At the end of the cycle, the host cell is lysed and infectious forms are produced. We analyse the mechanisms used by the bacteria to enter the cells, grow and modify them.
During the Chlamydia development cycle, the volume of the inclusions increases considerably, until they occupy a large portion of the cytoplasm. The inclusion membrane contains lipids that come from the host-cell. It also contains proteins produced by the bacteria which proliferate inside the inclusion. We have shown that Chlamydia use a secretion mechanism, which in other bacterial pathogen is involved in delivery of bacterial proteins within or through the membrane of eukaryotic host-cells. Furthermore, we have identified proteins which reach the inclusion membrane in this way. These proteins are very likely important in Chlamydia pathogenicity.
Finally, infection induces apoptosis (programmed cell death) of host cells in culture as well as in infected animals. This apoptosis, resulting from infection, takes place at the end of the infection cycle, by a mechanism independent of the usual effectors, the caspases. When apoptosis is inhibited, the infection yield is decreased suggesting that apoptosis is involved in the way bacteria exit infected cells at the end of the infectious cycle
Figure 1 : T lymphocyte (Tc) interacting with a cell presenting a bacterial superantigen (APC). The actin cytoskeleton polarisation is observed by confocal microscopy (left). On the right, the morphology of the two cells is seen by interferential contrast microscopy.
Figure 2 : Scheme of the infectious cycle of Chlamydia. The whole cycle takes place in the host-cell, in 48 to 72 hours. The infectious form of Chlamydia (EB) differentiates once inside the cell in the proliferative form (RB) which multiply and differentiate to EB at the end of the cycle.