Brazilian National Laboratory for Biosciences LNBio

Campinas, Brazil

Brazilian National Laboratory for Biosciences LNBio

Campinas, Brazil

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da Mata Madeira P.V.,Brazilian National Laboratory for Biosciences LNBio | Zouhir S.,Brazilian National Laboratory for Biosciences LNBio | Basso P.,Institut Universitaire de France | Basso P.,French National Center for Scientific Research | And 17 more authors.
Journal of Molecular Biology | Year: 2016

The type V secretion system is a macromolecular machine employed by a number of bacteria to secrete virulence factors into the environment. The human pathogen Pseudomonas aeruginosa employs the newly described type Vd secretion system to secrete a soluble variant of PlpD, a lipase of the patatin-like family synthesized as a single macromolecule that also carries a polypeptide transport-associated domain and a 16-stranded β-barrel. Here we report the crystal structure of the secreted form of PlpD in its biologically active state. PlpD displays a classical lipase α/β hydrolase fold with a catalytic site located within a highly hydrophobic channel that entraps a lipidic molecule. The active site is covered by a flexible lid, as in other lipases, indicating that this region in PlpD must modify its conformation in order for catalysis at the water-lipid interface to occur. PlpD displays phospholipase A1 activity and is able to recognize a number of phosphatidylinositols and other phosphatidyl analogs. PlpD is the first example of an active phospholipase secreted through the type V secretion system, for which there are more than 200 homologs, revealing details of the lipid destruction arsenal expressed by P. aeruginosa in order to establish infection. © 2016 Elsevier Ltd.


Favini-Stabile S.,CNRS Institute of Pharmacology and Structural Biology | Favini-Stabile S.,CEA Grenoble | Favini-Stabile S.,French National Center for Scientific Research | Contreras-Martel C.,CNRS Institute of Pharmacology and Structural Biology | And 9 more authors.
Environmental Microbiology | Year: 2013

Peptidoglycan is a major determinant of cell shape in bacteria, and its biosynthesis involves the concerted action of cytoplasmic, membrane-associated and periplasmic enzymes. Within the cytoplasm, Mur enzymes catalyse the first steps leading to peptidoglycan precursor biosynthesis, and have been suggested as being part of a multicomponent complex that could also involve the transglycosylase MurG and the cytoskeletal protein MreB. In order to initialize the characterization of a potential Mur interaction network, we purified MurD, MurE, MurF, MurG and MreB from Thermotoga maritima and characterized their interactions using membrane blotting and surface plasmon resonance. MurD, MurE and MurF all recognize MurG and MreB, but not each other, while the two latter proteins interact. In addition, we solved the crystal structures of MurD, MurE and MurF, which indicate that their C-termini display high conformational flexibilities. The differences in Mur conformations could be important parameters for the stability of an intracytoplasmic murein biosynthesis complex. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.


Neves D.,Brazilian National Laboratory for Biosciences LNBio | Job V.,CNRS Institute of Pharmacology and Structural Biology | Job V.,French Atomic Energy Commission | Job V.,French National Center for Scientific Research | And 10 more authors.
Journal of Molecular Biology | Year: 2013

Listeria monocytogenes is a human pathogen that employs a wide variety of virulence factors in order to adhere to, invade, and replicate within target cells. Internalins play key roles in processes ranging from adhesion to receptor recognition and are thus essential for infection. Recently, InlK, a surface-associated internalin, was shown to be involved in Listeria's ability to escape from autophagy by recruitment of the major vault protein (MVP) to the bacterial surface. Here, we report the structure of InlK, which harbors four domains arranged in the shape of a "bent arm". The structure supports a role for the "elbow" of InlK in partner recognition, as well as of two Ig-like pedestals intercalated by hinge regions in the projection of InlK away from the surface of the bacterium. The unusual fold and flexibility of InlK could be essential for MVP binding and concealment from recognition by molecules involved in the autophagic process. © 2013. Published by Elsevier Ltd. All rights reserved.


Tosi T.,CNRS Institute of Pharmacology and Structural Biology | Tosi T.,French National Center for Scientific Research | Tosi T.,CEA Grenoble | Estrozi L.F.,CNRS Institute of Pharmacology and Structural Biology | And 16 more authors.
Structure | Year: 2014

Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly. © 2014 Elsevier Ltd.


Shaik M.M.,CNRS Institute of Pharmacology and Structural Biology | Shaik M.M.,CEA Grenoble | Shaik M.M.,French National Center for Scientific Research | Maccagni A.,CNRS Institute of Pharmacology and Structural Biology | And 12 more authors.
Journal of Biological Chemistry | Year: 2014

Pili are surface-attached, fibrous virulence factors that play key roles in the pathogenesis process of a number of bacterial agents. Streptococcus pneumoniae is a causative agent of pneumonia and meningitis, and the appearance of drug-resistance organisms has made its treatment challenging, especially in developing countries. Pneumococcus-expressed pili are composed of three structural proteins: RrgB, which forms the polymerized backbone, RrgA, the tip-associated adhesin, and RrgC, which presumably associates the pilus with the bacterial cell wall. Despite the fact that the structures of both RrgA and RrgB were known previously, structural information for RrgC was still lacking, impeding the analysis of a complete model of pilus architecture. Here, we report the structure of RrgC to 1.85 Å and reveal that it is a three-domain molecule stabilized by two intradomain isopeptide bonds. RrgC does not depend on pilus-specific sortases to become attached to the cell wall; instead, it binds the preformed pilus to the peptidoglycan by employing the catalytic activity of SrtA. A comprehensive model of the type 1 pilus from S. pneumoniae is also presented. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.


Nikolaidis I.,CNRS Institute of Pharmacology and Structural Biology | Nikolaidis I.,CEA Grenoble | Nikolaidis I.,French National Center for Scientific Research | Nikolaidis I.,University Utrecht | And 7 more authors.
Protein Science | Year: 2014

Peptidoglycan is the main component of the bacterial cell wall. It is a complex, threedimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units crosslinked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies toward the development of novel inhibitors that could overcome resistance are also discussed. © 2014 The Protein Society.


Tosi T.,CNRS Institute of Pharmacology and Structural Biology | Tosi T.,CEA Grenoble | Tosi T.,French National Center for Scientific Research | Pflug A.,CNRS Institute of Pharmacology and Structural Biology | And 10 more authors.
Research in Microbiology | Year: 2013

Type III secretion systems (T3SS) are macromolecular complexes that translocate a wide number of effector proteins into eukaryotic host cells. Once within the cytoplasm, many T3SS effectors mimic the structure and/or function of eukaryotic proteins in order to manipulate signaling cascades, and thus play pivotal roles in colonization, invasion, survival and virulence. Structural biology techniques have played key roles in the unraveling of bacterial strategies employed for mimicry and targeting. This review provides an overall view of our current understanding of structure and function of T3SS effectors, as well as of the different classes of eukaryotic proteins that are targeted and the consequences for the infected cell. © 2013 Institut Pasteur.


Shaik M.M.,CNRS Institute of Pharmacology and Structural Biology | Shaik M.M.,French National Center for Scientific Research | Shaik M.M.,French Atomic Energy Commission | Lombardi C.,CNRS Institute of Pharmacology and Structural Biology | And 16 more authors.
Journal of Biological Chemistry | Year: 2015

Pili are fibrous appendages expressed on the surface of a vast number of bacterial species, and their role in surface adhesion is important for processes such as infection, colonization, and biofilm formation. The human pathogen Streptococcus pneumoniae expresses two different types of pili, PI-1 and PI-2, both of which require the concerted action of structural proteins and sortases for their polymerization. The type PI-1 streptococcal pilus is a complex, well studied structure, but the PI-2 type, present in a number of invasive pneumococcal serotypes, has to date remained less well understood. The PI-2 pilus consists of repeated units of a single protein, PitB, whose covalent association is catalyzed by cognate sortase SrtG-1 and partner protein SipA. Here we report the high resolution crystal structures of PitB and SrtG1 and use molecular modeling to visualize a "trapped" 1:1 complex between the two molecules. X-ray crystallography and electron microscopy reveal that the pneumococcal PI-2 backbone fiber is formed by PitB monomers associated in head-to-tail fashion and that short, flexible fibers can be formed even in the absence of coadjuvant proteins. These observations, obtained with a simple pilus biosynthetic system, are likely to be applicable to other fiber formation processes in a variety of Gram-positive organisms. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.


Wong S.G.,CNRS Institute of Pharmacology and Structural Biology | Wong S.G.,French National Center for Scientific Research | Wong S.G.,CEA Grenoble | Dessen A.,CNRS Institute of Pharmacology and Structural Biology | And 3 more authors.
Nature Communications | Year: 2014

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. Here we report the crystal structures and characterization of Salmonella enterica ser. Typhimurium A2M in different states of thioester activation. The structures reveal thirteen domains whose arrangement displays high similarity to proteins involved in eukaryotic immune defence. 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. © 2014 Macmillan Publishers Limited. All rights reserved.


PubMed | CNRS Institute of Pharmacology and Structural Biology and Brazilian National Laboratory for Biosciences LNBio
Type: Journal Article | Journal: Antibiotics (Basel, Switzerland) | Year: 2016

The bacterial cell wall is essential for survival, and proteins that participate in its biosynthesis have been the targets of antibiotic development efforts for decades. The biosynthesis of its main component, the peptidoglycan, involves the coordinated action of proteins that are involved in multi-member complexes which are essential for cell division (the divisome) and/or cell wall elongation (the elongasome), in the case of rod-shaped cells. Our knowledge regarding these interactions has greatly benefitted from the visualization of different aspects of the bacterial cell wall and its cytoskeleton by cryoelectron microscopy and tomography, as well as genetic and biochemical screens that have complemented information from high resolution crystal structures of protein complexes involved in divisome or elongasome formation. This review summarizes structural and functional aspects of protein complexes involved in the cytoplasmic and membrane-related steps of peptidoglycan biosynthesis, with a particular focus on protein-protein interactions whereby disruption could lead to the development of novel antibacterial strategies.

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