Institut de Biologie StructuraleGrenoble / France

Contact person(s) related to this article / CONTRERAS-MARTEL Carlos

Architecture and toxin secretion

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

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.

Fig.1.- Scheme of the type III secretion system of Pseudomonas aeruginosa highlighting structures solved by our group. (from Izoré, Job, and Dessen (2014) Structure 19, 603-612). Movie

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.

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 a/b 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 b-strand of ExoU in order to complete its 6-stranded b-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