Institut de Biologie StructuraleGrenoble / France

Contacts relatifs à cet article / CONTRERAS-MARTEL Carlos

Virulence factors and secretion systems

The T2SS secretin assembly mechanism

The type II secretion system plays a role in infection, colonization and microbial communication processes. An important element of many of these systems is the secretin, a membrane protein that forms a pore on the bacterial surface allowing toxins to be translocated towards the environment. Using cryo electron microscopy, crystallography, and microbial genetics techniques, and in collaboration with the MEM group of the IBS and the University of Saskatchewan in Canada, we recently solved the structures (at approximately 3.5 Å resolution) of two secretins from the emerging pathogens Vibrio vulnificus and Aeromonas hydrophila and characterized the mechanism for their assembly on the bacterial membrane. This work has shown that the assembly of some secretins requires the help of membrane lipo-proteins called "pilotins", while others are assembled independently. Since the secretin-pilotin interface is essential for the virulence of many pathogens, it could be an original target for the development of new inhibitors of infections caused by different bacteria.

Structure and assembly of pilotin-dependent and -independent secretins of the Type II secretion system. Howard SP, Estrozi L, Contreras-Martel C, Bertrand Q, Job V, Martins A, Schoehn G, Dessen A. PLoS Pathogens ; 15(5):e1007731

Bacteria possess a number of secretion systems whose goal is to transport toxins and effectors from the cytoplasm into the exterior environment, or into the cytoplasm of target cells. The type II secretion system (T2SS) consists of a dynamic assembly of 12-15 proteins that secrete, for example, multipartite holo-toxins and hydrolases such as pullulanase, through the outer membrane in their folded state. The type III secretion system (T3SS) has a membrane-embedded base and a hollow needle formed by a single polymerized protein through which effectors that manipulate host functions are translocated directly into the cytoplasm of target cells.

Architectural proteins : secretins

Tosi T, Estrozi LF, Job V, Guilvout I, Pugsley AP, Schoehn G, Dessen A. (2014) Structural similarity of secretins from type II and type III secretion systems. Structure 22, 1348-1355.

The outer membrane component of both T2SS and T3SS is the secretin, a protein that forms a homo-multimeric ring-like channel for needle assembly and toxin and hydrolase secretion. Our group, in close collaboration with the EM group headed by G. Schoehn at the IBS, has recently structurally characterized the secretins PulD (T2SS) from Klebsiella oxytoca and PscC (T3SS) from Pseudomonas aeruginosa, both of which were produced in a cell-free system, by cryo-EM and single particle analysis. Both structures display ‘cup and saucer’ architectures. These structures reveal internal details, such as a cavity at the level of the ‘saucer’ ring (that had not been observed in previously published structures), as well as a central plug and a periplasmic grid, that provide indications that the C-terminal portions of secretins consist of structural elements that can assume different conformations within the membrane.

Fig.2.- Three-dimensional cryo-EM reconstruction of the membrane-imbedded region of PscC from P. aeruginosa. Top, side, and bottom views shown at 1.3 sigma.

Fig. 3.- Cross-section view of PscC. The saggital density slice shows a cavity within the outer ring of the secretin, measuring approximately 70 Å, as well as a central plug and a constriction at the level of the periplasm.

Do bacteria have an immune system ?

Wong, S.G. and Dessen, A. (2014) Structure of a bacterial alpha-2-macroglobulin reveals mimicry of eukaryotic innate immunity. Nature Comm. 5, 4917

Alpha-2-macroglobulins (A2Ms) are plasma proteins that trap and inhibit a broad range of proteases and are major components of the eukaryotic innate immune system. Surprisingly, A2M-like proteins were identified in pathogenically invasive bacteria and species that colonize higher eukaryotes. Bacterial A2Ms are located in the periplasm where they are believed to provide protection to the cell by trapping external proteases through a covalent interaction with an activated thioester. This work reports the crystal structures and characterization of Salmonella enterica ser. Typhimurium A2M (Sa-A2M) in different states of thioester activation. The structures reveal thirteen domains whose arrangement displays high similarity to proteins involved in eukaryotic immune defense. A structural lock mechanism maintains the stability of the buried thioester, a requirement for its protease trapping activity. These findings indicate that bacteria have developed a rudimentary innate immune system whose mechanism mimics that of eukaryotes.

Collaborators : I. Attree, iRTSV Grenoble ; G. Schoehn, IBS Grenoble

Toxins : ExoU

Gendrin C, Contreras-Martel C, Bouillot S, Elsen S, Lemaire D, Skoufias DA, Huber P, Attree I, Dessen A (2012) Structural basis of cytotoxicity mediated by the type III secretion toxin ExoU from Pseudomonas aeruginosa. PLoS Pathog. e1002637.

ExoU is the most detrimental toxin injected by the T3SS of P. aeruginosa. ExoU is expressed by approximately 30% of clinical strains, 90% of which cause acute illness. It is encoded on a pathogenicity island together with its cognate chaperone SpcU, which is required for ExoU’s efficient secretion from the bacterial cytoplasm. ExoU is a 687-residue protein that, once translocated through the T3SS, induces cytotoxic effects leading to rapid necrotic cell death ; exoU knockout P. aeruginosa strains display greatly decreased virulence in mouse models of acute infection. In clinical settings, ExoU-expressing P. aeruginosa strains lead to poor patient prognosis, since the toxin causes acute lung epithelial injury and is linked to the development of septic shock.

Our work reveals the crystal structure of ExoU in a 1:1 complex with its chaperone, SpcU. ExoU folds into three distinct domains, which fulfill catalytic, bridging, and membrane-binding functions (Fig. 4). ExoU is a phospholipase, and thus its active site is localized deep within an alpha/beta hydrolase fold in a cleft sheltered by flexible loop regions.

Fig.4.- ExoU folds into three interconnected domains. SpcU, its cytosolic chaperone (green), clasps the initial beta-strand of ExoU in order to complete its 6-stranded beta-sheet.

Upon translocation into the eukaryotic cytoplasm, ExoU binds to the plasma membrane, where it is ubiquitinated on Lys178. We showed by bifluorescence complementation using the Venus fluorescent protein (Fig. 5) that Ub-ExoU not only localizes to the plasma membrane, but also co-localizes with markers of the endocytic pathway (Fig. 6) :

Fig.5.- HeLa cells transfected with clones expressing Ubiquitin and ExoU cloned downstream and upstream of N- and C-terminal domains of the Venus Fluorescent Protein reveal fluorescence not only on the cell membrane, but also within the cell

Fig.6.- ExoU co-localizes with typical markers of the endocytotic system, such as EEA1 (early endosome antigen 1) and lysotracker red (lysosome marker)

Despite the cell’s effort to destroy ExoU by shuttling it to lysosomes, it quickly dies as its bilayers get disrupted by ExoU’s potent phospholipase activity