Modular Architecture and Unique Teichoic Acid Recognition Features of Choline-Binding Protein L (CbpL) Contributing to Pneumococcal Pathogenesis

The human pathogen Streptococcus pneumoniae is decorated with a special class of surface-proteins known as choline-binding proteins (CBPs) attached to phosphorylcholine (PCho) moieties from cell-wall teichoic acids. By a combination of X-ray crystallography, NMR, molecular dynamics techniques and in vivo virulence and phagocytosis studies, we provide structural information of choline-binding protein L (CbpL) and demonstrate its impact on pneumococcal pathogenesis and immune evasion. CbpL is a very elongated three-module protein composed of (i) an Excalibur Ca 2+ -binding domain -reported in this work for the very first time-, (ii) an unprecedented anchorage module showing alternate disposition of canonical and non-canonical choline-binding sites that allows vine-like binding of fully-PCho-substituted teichoic acids (with two choline moieties per unit), and (iii) a Ltp-Lipoprotein domain. Our structural and infection assays indicate an important role of the whole multimodular protein allowing both to locate CbpL at specific places on the cell wall and to interact with host components in order to facilitate pneumococcal lung infection and transmigration from nasopharynx to the lungs and blood. CbpL implication in both resistance against killing by phagocytes and pneumococcal pathogenesis further postulate this surface-protein as relevant among the pathogenic arsenal of the pneumococcus.We gratefully acknowledge Karsta Barnekow and Kristine Sievert-Giermann, for technical assistance and Lothar Petruschka for in silico analysis (all Dept. of Genetics, University of Greifswald). We are further grateful to the staff from SLS synchrotron beamline for help in data collection. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG GRK 1870 (to SH) and the Spanish Ministry of Economy and Competitiveness (BFU2014-59389-P to JAH, CTQ2014-52633-P to MB and SAF2012-39760-C02-02 to FG) and S2010/BMD- 2457 (Community of Madrid to JAH and FG).Peer Reviewe

Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy

The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes

Viral infection modulation and neutralization by camelid nanobodies

International audienc

Structure and Functional Analysis of the Host Recognition Device of Lactococcal Phage Tuc2009

International audienc

J Biol Chem

beta-Barrel pore-forming toxins (beta-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a beta-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of beta-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other beta-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex

Human TR146 cells and pig buccal mucosa to assess oral transmucosal passage and buccal toxicity of foodgrade titanium dioxide

International audienceBackground: Today, the use of titanium dioxide (TiO 2) as food additive (E171) has been banned by the European Commission, due to concerns for human health based on studies showing TiO 2 particles systematically available, tissue accumulation, a genotoxic risk and possible promotion of precancerous lesions. However, E171 is still present in toothpastes and pharmaceutical tablets as a whitening powder mixing nano-and submicronic particles. Risk assessment of TiO 2 intake by oral route is mainly based on the assumption that particles are mainly absorbed by the intestine. However, while the buccal mucosa is the first exposed area, the possibility of an oro-transmucosal passage has not been documented so far. In order to gain insight on possible adverse effects for human health associated to E171 buccal exposure, we analyzed in vivo the translocation of TiO 2 (E171) in the buccal mucosa of pigs used as human mouth model. Moreover, we evaluated in vitro the particle translocation on human buccal TR146 cell line, and measured cytotoxic and genotoxic effects on proliferative and differentiated epithelial cells. Methods & Results: Under realistic exposure conditions with 50 µg/ml of food-grade TiO 2 in water suspension (size distribution 20-440 nm; mean size of 105 nm) deposited under the tongue of pigs, TEM-EDX data revealed the presence of small aggregates of TiO 2 particles translocated into the buccal mucosa from 30 minutes of exposure, reaching submaxillary lymph nodes after 4 hours. In human TR146 cells exposed to E171, kinetic analysis using confocal, TEM and SIMS imaging showed progressive and large uptake of isolated or small aggregates of both submicronic and nanosized particles, showing high permeability capacity. At 2h of E171 exposure, cytotoxicity, genotoxicity and oxidative stress were investigated on both proliferative or differentiated TR146 cells, in comparison with two TiO 2 size standards of 115 nm and 21 nm in diameter. All tested TiO 2 particles were reported cytotoxic on proliferative TR146 cells, and this effect was almost abolished following differentiation. Oxidative stress and genotoxicity assessed through γH2AX and 53BP1 foci formation and comet assay were only reported for E171 sample and TiO 2 particles of 115 nm, suggesting the particles above 20 nm responsive of these effects, and mainly on proliferative cells. Conclusions: Taken together, these results show in vivo and in vitro that the buccal mucosa is an important absorption route for systemic passage of foodborne TiO 2 (E171) particles. In human cells, uptake of TiO2 particles was cytotoxic without size effects, while they generate further oxidative and genotoxic stresses in proliferative buccal cells, that could impair epithelium renewal in the mouth. Altogether, these data emphasize that buccal exposure should be considered for toxicokinetic and risk assessments of TiO2 in human when used as food additive, including in toothpaste and pharmaceutical formulations