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142 results on '"Andréa Dessen"'

1. Self-association of MreC as a regulatory signal in bacterial cell wall elongation

Author: Alexandre Martins, Carlos Contreras-Martel, Manon Janet-Maitre, Mayara M. Miyachiro, Leandro F. Estrozi, Daniel Maragno Trindade, Caíque C. Malospirito, Fernanda Rodrigues-Costa, Lionel Imbert, Viviana Job, Guy Schoehn, Ina Attrée, and Andréa Dessen
Subjects: Science
Abstract: MreC is a membrane-associated protein that modulates the activity of the elongasome, a protein complex that controls cell wall formation in rod-shaped bacteria. Here, the authors use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC in atomic detail.
Published: 2021
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2. Draft genome sequence of Psychrobacter nivimaris LAMA 639 and its biotechnological potential

Author: Brendon Egon Kormann Staloch, Henrique Niero, Robert Cardoso de Freitas, Patricia Ballone, Fernanda Rodrigues-Costa, Daniela Barretto Barbosa Trivella, Andréa Dessen, Marcus Adonai Castro da Silva, and André Oliveira de Souza Lima
Subjects: Psychrobacter, Extremophile, Whole-genome sequencing, Illumina, Enzyme exploiting, Computer applications to medicine. Medical informatics, R858-859.7, Science (General), Q1-390
Abstract: Bacteria of the genus Psychrobacter are known for their psychrophilic characteristics, being extremophilic organisms capable of surviving and reproducing in hostile environments of low temperature and high pressure. Among many of the genus characteristics, there is the ability to produce enzymes and molecules of industrial biotechnology importance, such as pigments and proteins related to heavy metal bioremediation. The bacterium strain Psychrobacter nivimaris LAMA 639 was isolated from sediments from the Walvis Ridge ocean crest at a depth of 4.400 m (33.40 S 2.35 E). It is a nonmotile, halotolerant, cream-colored gram-negative aerobic bacterium. Its cultivation was performed in marine agar plates and inoculated into test tubes with NaCl at an optimal temperature of 30 °C and with shaking at 100 rpm. Genome extraction was performed with the DNeasy Blood & Tissue Kit (QIAGEN®). Sequencing was performed by Macrogen using the NovaSeq® 6000 platform (Illumina) applying the whole genome shotgun (WGS) method. Thereafter, 14.712.526 reads of 151 bp were generated, totaling 2.2 G bp with a GC content of 42.9%. Assembly and mapping were performed with a CLC Genomics Workbench. The best assembly considered was the one with the lowest number of contigs and the highest base length pair. The assemblies were evaluated using QUAST, and the best resulting variant was selected for annotation. Genome annotation was performed with RAST and PATRIC; the antiSMASH tool was used for secondary metabolites; NaPDoS was used for domains; and three-dimensional structural prediction of relevant proteins was performed using Phyre2. Annotation with ClassicRAST generated 2,891 coding sequences (CDSs) distributed in 402 subsystems. Annotation with PATRIC generated 2,896 coding sequences, among them 776 hypothetical proteins. The antiSMASH tool visualized a beta-lactone cluster in contig 06. In the search for natural products with NaPDoS, two ketosynthase domains were identified. The search for relevant proteins was performed using the AMFEP list as a criterion. From these data, 34 possible enzymes with biotechnological potential were found. Finally, the organism is presented as a new reference regarding the potential of deep-sea marine bacteria, demonstrating that, from the annotated and cured genome, it is possible to find in its genetic repertory products of interest for biotechnological applications.
Published: 2022
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3. The inherent flexibility of type I non-ribosomal peptide synthetase multienzymes drives their catalytic activities

Author: Sarah Bonhomme, Andréa Dessen, and Pauline Macheboeuf
Subjects: non-ribosomal peptide synthetases, flexibility, supramodular architecture, Biology (General), QH301-705.5
Abstract: Non-ribosomal peptide synthetases (NRPSs) are multienzymes that produce complex natural metabolites with many applications in medicine and agriculture. They are composed of numerous catalytic domains that elongate and chemically modify amino acid substrates or derivatives and of non-catalytic carrier protein domains that can tether and shuttle the growing products to the different catalytic domains. The intrinsic flexibility of NRPSs permits conformational rearrangements that are required to allow interactions between catalytic and carrier protein domains. Their large size coupled to this flexibility renders these multi-domain proteins very challenging for structural characterization. Here, we summarize recent studies that offer structural views of multi-domain NRPSs in various catalytically relevant conformations, thus providing an increased comprehension of their catalytic cycle. A better structural understanding of these multienzymes provides novel perspectives for their re-engineering to synthesize new bioactive metabolites.
Published: 2021
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4. Publisher Correction: Self-association of MreC as a regulatory signal in bacterial cell wall elongation

Author: Alexandre Martins, Carlos Contreras-Martel, Manon Janet-Maitre, Mayara M. Miyachiro, Leandro F. Estrozi, Daniel Maragno Trindade, Caíque C. Malospirito, Fernanda Rodrigues-Costa, Lionel Imbert, Viviana Job, Guy Schoehn, Ina Attrée, and Andréa Dessen
Subjects: Science
Published: 2022
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5. Molecular architecture of the PBP2–MreC core bacterial cell wall synthesis complex

Author: Carlos Contreras-Martel, Alexandre Martins, Chantal Ecobichon, Daniel Maragno Trindade, Pierre-Jean Matteï, Samia Hicham, Pierre Hardouin, Meriem El Ghachi, Ivo G. Boneca, and Andréa Dessen
Subjects: Science
Abstract: Bacterial wall biosynthesis is a complex process that requires the coordination of multiple enzymes. Here, the authors structurally characterize the PBP2:MreC complex involved in peptidoglycan elongation and cross-linking, and demonstrate that its disruption leads to loss of H. pylori shape and inability to sustain growth.
Published: 2017
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6. Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.

Author: S Peter Howard, Leandro F Estrozi, Quentin Bertrand, Carlos Contreras-Martel, Timothy Strozen, Viviana Job, Alexandre Martins, Daphna Fenel, Guy Schoehn, and Andréa Dessen
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.
Published: 2019
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7. Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa

Author: Charlotte Lombardi, James Tolchard, Stephanie Bouillot, Luca Signor, Caroline Gebus, David Liebl, Daphna Fenel, Jean-Marie Teulon, Juliane Brock, Birgit Habenstein, Jean-Luc Pellequer, Eric Faudry, Antoine Loquet, Ina Attrée, Andréa Dessen, and Viviana Job
Subjects: type III secretion system, Pseudomonas aeruginosa, T3SS needle, structure, virulence, immunofluorescence microscopy, Microbiology, QR1-502
Abstract: The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo, in vitro, and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro. These results suggest that the T3SS needle of P. aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.
Published: 2019
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8. Chemical Elicitors Induce Rare Bioactive Secondary Metabolites in Deep-Sea Bacteria under Laboratory Conditions

Author: Rafael de Felício, Patricia Ballone, Cristina Freitas Bazzano, Luiz F. G. Alves, Renata Sigrist, Gina Polo Infante, Henrique Niero, Fernanda Rodrigues-Costa, Arthur Zanetti Nunes Fernandes, Luciane A. C. Tonon, Luciana S. Paradela, Renna Karoline Eloi Costa, Sandra Martha Gomes Dias, Andréa Dessen, Guilherme P. Telles, Marcus Adonai Castro da Silva, Andre Oliveira de Souza Lima, and Daniela Barretto Barbosa Trivella
Subjects: bacterial natural products, natural product libraries, drug discovery, chemical elicitors, cryptic gene clusters, chemical space, Microbiology, QR1-502
Abstract: Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.
Published: 2021
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9. Crystallographic Study of Peptidoglycan Biosynthesis Enzyme MurD: Domain Movement Revisited.

Author: Roman Šink, Miha Kotnik, Anamarija Zega, Hélène Barreteau, Stanislav Gobec, Didier Blanot, Andréa Dessen, and Carlos Contreras-Martel
Subjects: Medicine, Science
Abstract: The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-MurNAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway.
Published: 2016
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10. Structural Basis for the Inhibition of the Chromatin Repressor BAHD1 by the Bacterial Nucleomodulin LntA

Author: Alice Lebreton, Viviana Job, Marie Ragon, Alban Le Monnier, Andréa Dessen, Pascale Cossart, and Hélène Bierne
Subjects: Microbiology, QR1-502
Abstract: ABSTRACT The nucleus has emerged as a key target for nucleomodulins, a family of effectors produced by bacterial pathogens to control host transcription or other nuclear processes. The virulence factor LntA from Listeria monocytogenes stimulates interferon responses during infection by inhibiting BAHD1, a nuclear protein involved in gene silencing by promoting heterochromatin formation. So far, whether the interaction between LntA and BAHD1 is direct and sufficient for inhibiting BAHD1 activity is unknown. Here, we functionally characterized the molecular interface between the two proteins in vitro and in transfected or infected human cells. Based on the known tridimensional structure of LntA, we identified a dilysine motif (K180/K181) in the elbow region of LntA and a central proline-rich region in BAHD1 as crucial for the direct LntA-BAHD1 interaction. To better understand the role played by the dilysine motif in the functionality of LntA, we solved the crystal structure of a K180D/K181D mutant to a 2.2-Å resolution. This mutant highlights a drastic redistribution of surface charges in the vicinity of a groove, which likely plays a role in nucleomodulin target recognition. Mutation of the strategic dilysine motif also abolished the recruitment of LntA to BAHD1-associated nuclear foci and impaired the LntA-mediated stimulation of interferon responses upon infection. Last, the strict conservation of residues K180 and K181 in LntA sequences from 188 L. monocytogenes strains of different serotypes and origins further supports their functional importance. Together, these results provide structural and functional details about the mechanism of inhibition of an epigenetic factor by a bacterial nucleomodulin. IMPORTANCE Pathogens have evolved various strategies to deregulate the expression of host defense genes during infection, such as targeting nuclear proteins. LntA, a secreted virulence factor from the bacterium Listeria monocytogenes, stimulates innate immune responses by inhibiting a chromatin-associated repressor, BAHD1. This study reveals the structural features of LntA required for BAHD1 inhibition. LntA interacts directly with a central domain of BAHD1 via a surface patch of conserved positive charges, located nearby a groove on the elbow region of LntA. By demonstrating that this patch is required for LntA function, we provide a better understanding of the molecular mechanism allowing a bacterial pathogen to control host chromatin compaction and gene expression.
Published: 2014
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11. Unique Features of a Pseudomonas aeruginosa α2-Macroglobulin Homolog

Author: Mylène Robert-Genthon, Maria Guillermina Casabona, David Neves, Yohann Couté, Félix Cicéron, Sylvie Elsen, Andréa Dessen, and Ina Attrée
Subjects: Microbiology, QR1-502
Abstract: ABSTRACT Human pathogens frequently use protein mimicry to manipulate host cells in order to promote their survival. Here we show that the opportunistic pathogen Pseudomonas aeruginosa synthesizes a structural homolog of the human α2-macroglobulin, a large-spectrum protease inhibitor and important player of innate immunity. Small-angle X-ray scattering analysis demonstrated that the fold of P. aeruginosa MagD (PA4489) is similar to that of the human macroglobulin and undergoes a conformational modification upon binding of human neutrophil elastase. MagD synthesis is under the control of a general virulence regulatory pathway including the inner membrane sensor RetS and the RNA-binding protein RsmA, and MagD undergoes cleavage from a 165-kDa to a 100-kDa form in all clinical isolates tested. Fractionation and immunoprecipitation experiments showed that MagD is translocated to the bacterial periplasm and resides within the inner membrane in a complex with three other molecular partners, MagA, MagB, and MagF, all of them encoded by the same six-gene genetic element. Inactivation of the whole 10-kb operon on the PAO1 genome resulted in mislocalization of uncleaved, in trans-provided MagD as well as its rapid degradation. Thus, pathogenic bacteria have acquired a homolog of human macroglobulin that plays roles in host-pathogen interactions potentially through recognition of host proteases and/or antimicrobial peptides; it is thus essential for bacterial defense. IMPORTANCE The pathogenesis of Pseudomonas aeruginosa is multifactorial and relies on surface-associated and secreted proteins with different toxic activities. Here we show that the bacterium synthesizes a 160-kDa structural homolog of the human large-spectrum protease inhibitor α2-macroglobulin. The bacterial protein is localized in the periplasm and is associated with the inner membrane through the formation of a multimolecular complex. Its synthesis is coregulated at the posttranscriptional level with other virulence determinants, suggesting that it has a role in bacterial pathogenicity and/or in defense against the host immune system. Thus, this new P. aeruginosa macromolecular complex may represent a future target for antibacterial developments.
Published: 2013
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12. Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis

Author: Federica Laddomada, Mayara M. Miyachiro, and Andréa Dessen
Subjects: peptidoglycan, elongation, cell division, protein complexes, Mur enzymes, MraY, bacterial cytoskeleton, Therapeutics. Pharmacology, RM1-950
Abstract: 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.
Published: 2016
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13. Structural basis of cytotoxicity mediated by the type III secretion toxin ExoU from Pseudomonas aeruginosa.

Author: Claire Gendrin, Carlos Contreras-Martel, Stéphanie Bouillot, Sylvie Elsen, David Lemaire, Dimitrios A Skoufias, Philippe Huber, Ina Attree, and Andréa Dessen
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P₂. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.
Published: 2012
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14. Conformational states of a bacterial α2-macroglobulin resemble those of human complement C3.

Author: David Neves, Leandro F Estrozi, Viviana Job, Frank Gabel, Guy Schoehn, and Andréa Dessen
Subjects: Medicine, Science
Abstract: α(2) macroglobulins (α(2)Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α(2)Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α(2)M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α(2)M homolog (ECAM) was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS) techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials.
Published: 2012
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15. Hijacking of the pleiotropic cytokine interferon-γ by the type III secretion system of Yersinia pestis.

Author: Claire Gendrin, Stéphane Sarrazin, David Bonnaffé, Jean-Michel Jault, Hugues Lortat-Jacob, and Andréa Dessen
Subjects: Medicine, Science
Abstract: Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague.
Published: 2010
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16. Architecture of a PKS-NRPS hybrid megaenzyme involved in the biosynthesis of the genotoxin colibactin

Author: Sarah Bonhomme, Carlos Contreras-Martel, Andréa Dessen, Pauline Macheboeuf, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)
Subjects: MESH: Polyketide Synthases, Structural Biology, MESH: Escherichia coli, [SDV]Life Sciences [q-bio], Non-ribosomal peptide synthetase, MESH: X-Ray Diffraction, Colibactin, Polyketide synthase, Molecular Biology, MESH: Scattering, Small Angle
Abstract: International audience; The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications.
Published: 2023
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17. Architecture and genomic arrangement of the MurE–MurF bacterial cell wall biosynthesis complex

Author: Karina T. Shirakawa, Fernanda Angélica Sala, Mayara M. Miyachiro, Viviana Job, Daniel Maragno Trindade, Andréa Dessen, Brazilian National Laboratory for Biosciences (LNBio), Centro Nacional de Pesquisa em Energia e materiais, Centra Nacional de Pesquisa-Centra Nacional de Pesquisa, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)
Subjects: Bordetella spp, MESH: Bacteria, MESH: Cell Wall, Multidisciplinary, chimeric proteins, MESH: Ligases, MESH: Peptidoglycan, MESH: Genomics, [SDV]Life Sciences [q-bio], peptidoglycan, MESH: Peptide Synthases, Mur ligases, MESH: Bacterial Proteins
Abstract: Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE–MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE–MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE–MurF interacts with other Mur ligases via its central domains with K D s in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.
Published: 2023
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18. Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with β-Lactones

Author: Parker L. Flanders, Carlos Contreras-Martel, Nathaniel W. Brown, Joshua D. Shirley, Alexandre Martins, Kelsie N. Nauta, Andréa Dessen, Erin E. Carlson, Elizabeth A. Ambrose, University of Minnesota, Minneapolis, USA, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Grenoble Instruct-ERIC center (ISBG, UAR 3518 CNRS-CEA-UGA-EMBL), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010)
Subjects: MESH: Penicillin-Binding Proteins, [SDV]Life Sciences [q-bio], General Medicine, Molecular Dynamics Simulation, beta-Lactams, Ligands, Biochemistry, Article, Anti-Bacterial Agents, Lactones, Streptococcus pneumoniae, Bacterial Proteins, MESH: beta-Lactams, MESH: Anti-Bacterial Agents, MESH: Ligands, Penicillin-Binding Proteins, Molecular Medicine, MESH: Molecular Dynamics Simulation, MESH: Bacterial Proteins, MESH: Lactones, MESH: Streptococcus pneumoniae
Abstract: International audience; β-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to β-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of β-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained
Published: 2022
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19. Draft genome sequence of

Author: Brendon Egon Kormann, Staloch, Henrique, Niero, Robert Cardoso de, Freitas, Patricia, Ballone, Fernanda, Rodrigues-Costa, Daniela Barretto Barbosa, Trivella, Andréa, Dessen, Marcus Adonai Castro da, Silva, and André Oliveira de Souza, Lima
Abstract: Bacteria of the genus
Published: 2021

20. The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis

Author: Delphine Patin, Dominique Mengin-Lecreulx, Andréa Dessen, Cécile Breyton, Mayara M. Miyachiro, Christine Ebel, Federica Laddomada, Aline Le Roy, Matthew Jessop, Irina Gutsche, Viviana Job, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), AUC, ME, biophysique, ANR-13-BSV8-0015,BACSCREEN,Assemblage et structure de complexes macromoléculaires essentiels pour la biosynthèse de la paroi bactérienne(2013), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0301 basic medicine, Cytoplasm, Operon, lcsh:Medicine, Peptidoglycan, N-Acetylglucosaminyltransferases, Bordetella pertussis, Article, Bacterial cell structure, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Bacterial Proteins, X-Ray Diffraction, Biosynthesis, Cell Wall, Catalytic Domain, Scattering, Small Angle, Glycosyltransferase, Humans, Inner membrane, Peptide Synthases, lcsh:Science, chemistry.chemical_classification, Binding Sites, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, lcsh:R, Glycosyltransferases, Anti-Bacterial Agents, Cell biology, 030104 developmental biology, Enzyme, chemistry, biology.protein, lcsh:Q, 030217 neurology & neurosurgery, Bacterial Outer Membrane Proteins, Protein Binding
Abstract: Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE–MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE–MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.
Published: 2019
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21. MagC is a NplC/P60‐like member of the $\alpha$‐2‐macroglobulin Mag complex of $Pseudomonas\ aeruginosa$ that interacts with peptidoglycan

Author: Ina Attrée, Andréa Dessen, Daniel M. Trindade, Carlos Contreras-Martel, Pauline Macheboeuf, Samira Zouhir, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Pathogenèse bactérienne et réponses cellulaires (PBRC), Centre National de la Recherche Scientifique (CNRS)-Biologie du Cancer et de l'Infection (BCI ), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Groupe Pathogenèse Bactérienne et Réponses Cellulaires / Bacterial Pathogenesis and Cellular Responses Group (IBS-PBRC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Operon, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Biophysics, Peptidoglycan, Calorimetry, medicine.disease_cause, Crystallography, X-Ray, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, Genetics, medicine, Amino Acid Sequence, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Protease, Sequence Homology, Amino Acid, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Pseudomonas aeruginosa, 030302 biochemistry & molecular biology, Isothermal titration calorimetry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Pregnancy-Associated alpha 2-Macroglobulins, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Macroglobulin, chemistry, Macroglobulins, Ultracentrifugation, Function (biology), Protein Binding
Abstract: International audience; Bacterial $\alpha$-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In $Pseudomonas\ aeruginosa$, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.
Published: 2021
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22. The inherent flexibility of type I non-ribosomal peptide synthetase multienzymes drives their catalytic activities

Author: Pauline Macheboeuf, Sarah Bonhomme, Andréa Dessen, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)
Subjects: QH301-705.5, Immunology, Peptide Synthetases, Peptide, Computational biology, Review, Biology, 01 natural sciences, General Biochemistry, Genetics and Molecular Biology, Catalysis, 03 medical and health sciences, Structure-Activity Relationship, Catalytic Domain, supramodular architecture, Biology (General), Peptide Synthases, Review Articles, 030304 developmental biology, chemistry.chemical_classification, Flexibility (engineering), 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], non-ribosomal peptide synthetases, 010405 organic chemistry, General Neuroscience, Ribosomal RNA, 0104 chemical sciences, 3. Good health, Amino acid, flexibility, chemistry, Catalytic cycle, Carrier protein
Abstract: International audience; Non-ribosomal peptide synthetases (NRPSs) are multienzymes that produce complex natural metabolites with many applications in medicine and agriculture. They are composed of numerous catalytic domains that elongate and chemically modify amino acid substrates or derivatives and of non-catalytic carrier protein domains that can tether and shuttle the growing products to the different catalytic domains. The intrinsic flexibility of NRPSs permits conformational rearrangements that are required to allow interactions between catalytic and carrier protein domains. Their large size coupled to this flexibility renders these multi-domain proteins very challenging for structural characterization. Here, we summarize recent studies that offer structural views of multi-domain NRPSs in various catalytically relevant conformations, thus providing an increased comprehension of their catalytic cycle. A better structural understanding of these multienzymes provides novel perspectives for their re-engineering to synthesize new bioactive metabolites.
Published: 2021
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23. Author response for 'The inherent flexibility of type I non-ribosomal peptide synthetase multienzymes drives their catalytic activities'

Author: Andréa Dessen, Sarah Bonhomme, and Pauline Macheboeuf
Subjects: chemistry.chemical_classification, Flexibility (engineering), chemistry, Biochemistry, Peptide, Ribosomal RNA
Published: 2021
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24. Self-association of MreC as a regulatory signal in bacterial cell wall elongation

Author: Carlos Contreras-Martel, Leandro F. Estrozi, Daniel M. Trindade, Mayara M. Miyachiro, Fernanda Rodrigues-Costa, Ina Attrée, Guy Schoehn, Caíque C. Malospirito, Alexandre Martins, Viviana Job, Andréa Dessen, Lionel Imbert, Manon Janet-Maitre, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Groupe Pathogenèse Bactérienne et Réponses Cellulaires / Bacterial Pathogenesis and Cellular Responses Group (IBS-PBRC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Groupe de RMN biomoléculaire (IBS-NMR), and ANR-18-CE11-0019,PSEUDO-WALL,Architecture et fonction de complexes de la biosynthèse de la paroi cellulaire de Pseudomonas aeruginosa(2018)
Subjects: 0301 basic medicine, Science, General Physics and Astronomy, MESH: Amino Acid Sequence, MreB, General Biochemistry, Genetics and Molecular Biology, Bacterial cell structure, Article, MESH: Recombinant Proteins, 03 medical and health sciences, 0302 clinical medicine, Protein structure, MESH: Cell Wall, Phylogenetics, Cryoelectron microscopy, MESH: Phylogeny, MESH: Bacterial Proteins, MESH: Mutagenesis, X-ray crystallography, chemistry.chemical_classification, MESH: Protein Conformation, alpha-Helical, Bacterial structural biology, Multidisciplinary, MESH: Conserved Sequence, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, MESH: Protein Multimerization, Mutagenesis, General Chemistry, biology.organism_classification, MESH: Crystallography, X-Ray, Cell biology, 030104 developmental biology, Enzyme, MESH: Pseudomonas aeruginosa, MESH: Protein Domains, MESH: Protein Conformation, beta-Strand, MESH: Cryoelectron Microscopy, Elongation, 030217 neurology & neurosurgery, Bacteria
Abstract: The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC’s ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity., MreC is a membrane-associated protein that modulates the activity of the elongasome, a protein complex that controls cell wall formation in rod-shaped bacteria. Here, the authors use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC in atomic detail.
Published: 2021
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25. Chemical Elicitors Induce Rare Bioactive Secondary Metabolites in Deep-Sea Bacteria under Laboratory Conditions

Author: Sandra M. G. Dias, Luiz F. G. Alves, Gina Polo Infante, Fernanda Rodrigues-Costa, Renna Karoline Eloi Costa, Cristina Freitas Bazzano, Arthur Zanetti Nunes Fernandes, Luciana S. Paradela, Renata Sigrist, Guilherme P. Telles, André Oliveira de Souza Lima, Rafael de Felício, Marcus Adonai Castro da Silva, Luciane Alessandra Chimetto Tonon, Daniela B. B. Trivella, Patricia Ballone, Henrique Niero, Andréa Dessen, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institute of Computing [Campinas] (IC), Universidade Estadual de Campinas = University of Campinas (UNICAMP), School of Sea, Science and Technology, Universidade do Vale do Itajaí (UNIVALI), Institute of Computing [Campinas] (UNICAMP), and Universidade Estadual de Campinas (UNICAMP)
Subjects: 0301 basic medicine, Endocrinology, Diabetes and Metabolism, lcsh:QR1-502, Bacterial genome size, 01 natural sciences, Biochemistry, lcsh:Microbiology, Article, drug discovery, 03 medical and health sciences, chemistry.chemical_compound, Marine bacteriophage, Molecular Biology, bacterial natural products, natural product libraries, chemical elicitors, cryptic gene clusters, chemical space, molecular networking, dereplication, deep-sea bacteria, LC-MS/MS data mining, Natural product, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010405 organic chemistry, Drug discovery, Chemistry, Biological activity, biology.organism_classification, Chemical space, 0104 chemical sciences, 030104 developmental biology, Cheminformatics, Bacteria
Abstract: International audience; Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.
Published: 2021
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26. Merulinic acid C overcomes gentamicin resistance in Enterococcus faecium

Author: Frederico J. Gueiros-Filho, Ana C.G. Cauz, Daniela B. B. Trivella, Marcelo Brocchi, Vítor F. Freire, Ariane F. Bertonha, Paulo Vinicius da Mata Madeira, Fernanda Rodrigues-Costa, Andréa Dessen, Daniel M. Trindade, Antonio G. Ferreira, Marcos Guilherme da Cunha, Laura P. Ióca, Ana Cristina Gales, Cristina Dislich Ropke, Roberto G. S. Berlinck, Rafael de Felício, Juliano Slivinski, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Unité fonctionnelle d'épilepsie [CHU Pitié-Salpêtrière], Service de Neurologie [CHU Pitié-Salpêtrière], IFR70-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-IFR70-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Instituto de Biologia [Campinas], Universidade Estadual de Campinas (UNICAMP), British Geological Survey (BGS), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-IFR70-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), and Universidade Estadual de Campinas = University of Campinas (UNICAMP)
Subjects: medicine.drug_class, Enterococcus faecium, Antibiotics, ved/biology.organism_classification_rank.species, 01 natural sciences, Biochemistry, Gentamicin resistance, Microbiology, chemistry.chemical_compound, Drug Resistance, Bacterial, Drug Discovery, Hydroxybenzoates, medicine, Humans, Model organism, Molecular Biology, Gram-Positive Bacterial Infections, Natural product, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 010405 organic chemistry, ved/biology, Organic Chemistry, Drug Synergism, biology.organism_classification, Anti-Bacterial Agents, 3. Good health, 0104 chemical sciences, Anacardic acids, 010404 medicinal & biomolecular chemistry, chemistry, Gentamicin, Peptidoglycan, Gentamicins, medicine.drug, PRODUTOS NATURAIS
Abstract: International audience; Enterococci are gram-positive, widespread nosocomial pathogens that in recent years have developed resistance to various commonly employed antibiotics. Since finding new infection-control agents based on secondary metabolites from organisms has proved successful for decades, natural products are potentially useful sources of compounds with activity against enterococci. Herein are reported the results of a natural product library screening based on a whole-cell assay against a gram-positive model organism, which led to the isolation of a series of anacardic acids identified by analysis of their spectroscopic data and by chemical derivatizations. Merulinic acid C was identified as the most active anacardic acid derivative obtained against antibiotic-resistant enterococci. Fluorescence microscopy analyses showed that merulinic acid C targets the bacterial membrane without affecting the peptidoglycan and causes rapid cellular ATP leakage from cells. Merulinic acid C was shown to be synergistic with gentamicin against Enterococcus faecium, indicating that this compound could inspire the development of new antibiotic combinations effective against drug-resistant pathogens.
Published: 2020
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27. Bacterial secretins: Mechanisms of assembly and membrane targeting

Author: Yuri Rafael de Oliveira Silva, Carlos Contreras-Martel, Andréa Dessen, Pauline Macheboeuf, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)
Subjects: Models, Molecular, Cryo-electron microscopy, protein-protein interactions, Virulence, Reviews, Type IV pilus system, Biochemistry, secretin, Pilus, Protein–protein interaction, Bacterial genetics, 03 medical and health sciences, Secretion, Types II and III secretion systems, Molecular Biology, 030304 developmental biology, 0303 health sciences, Bacteria, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Effector, bacterial virulence, 030302 biochemistry & molecular biology, Cryoelectron Microscopy, Cell biology, Cytoplasm, toxin secretion, Bacterial Outer Membrane Proteins
Abstract: International audience; Secretion systems are employed by bacteria to transport macromolecules across membranes without compromising their integrities. Processes including virulence, colonization, and motility are highly dependent on the secretion of effector molecules toward the immediate cellular environment, and in some cases, into the host cytoplasm. In Type II and Type III secretion systems, as well as in Type IV pili, homomultimeric complexes known as secretins form large pores in the outer bacterial membrane, and the localization and assembly of such 1 MDa molecules often relies on pilotins or accessory proteins. Significant progress has been made toward understanding details of interactions between secretins and their partner proteins using approaches ranging from bacterial genetics to cryo electron microscopy. This review provides an overview of the mode of action of pilotins and accessory proteins for T2SS, T3SS, and T4PS secretins, highlighting recent near-atomic resolution cryo-EM secretin complex structures and underlining the importance of these interactions for secretin functionality.
Published: 2020
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28. Penicillin-Binding Proteins (PBPs) and Bacterial Cell Wall Elongation Complexes

Author: Mayara M, Miyachiro, Carlos, Contreras-Martel, and Andréa, Dessen
Subjects: Bacteria, Cell Wall, Penicillin-Binding Proteins, Peptidoglycan
Abstract: The bacterial cell wall is the validated target of mainstream antimicrobials such as penicillin and vancomycin. Penicillin and other β-lactams act by targeting Penicillin-Binding Proteins (PBPs), enzymes that play key roles in the biosynthesis of the main component of the cell wall, the peptidoglycan. Despite the spread of resistance towards these drugs, the bacterial cell wall continues to be a major Achilles' heel for microbial survival, and the exploration of the cell wall formation machinery is a vast field of work that can lead to the development of novel exciting therapies. The sheer complexity of the cell wall formation process, however, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. New developments in genetic and biochemical screens, as well as different aspects of structural biology, have shed new light on the importance of complexes formed by PBPs, notably within the cell wall elongation machinery. This chapter summarizes structural and functional details of PBP complexes involved in the periplasmic and membrane steps of peptidoglycan biosynthesis with a focus on cell wall elongation. These assemblies could represent interesting new targets for the eventual development of original antibacterials.
Published: 2020

29. An RNA-Binding Protein Secreted by a Bacterial Pathogen Modulates RIG-I Signaling

Author: Alessandro Pagliuso, Bruno Dupuy, Christophe Bécavin, Mikael Koutero, Alice Lebreton, Frédéric Tangy, Andréa Dessen, Marie-Anne Nahori, Valérie Najburg, Fabrizia Stavru, Eric Allemand, To Nam Tham, Pascale Cossart, Anastassia V. Komarova, Christian Muchardt, Stevens Robertin, Sergey Bessonov, Quentin Bertrand, Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Régulation épigénétique - Epigenetic regulation, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Pathogénèse des Bactéries Anaérobies / Pathogenesis of Bacterial Anaerobes (PBA (U-Pasteur_6)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Génomique virale et vaccination, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Brazilian National Laboratory for Biosciences (LNBio), Centro Nacional de Pesquisa em Energia e materiais, Centra Nacional de Pesquisa-Centra Nacional de Pesquisa, Institut National de la Recherche Agronomique (INRA), Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA). We are grateful to R.Y. Sanchez David and R. Viber for technical support. Work in A.L.’s team was supported by Inserm ATIP-Avenir and Mairie de Paris (Programme Émergences—Recherche médicale). This work was supported by grants from the European Research Council (ERC) advanced grant BacCellEpi (670823) and the Fondation Le Roch to P.C., We thank all members of the Cossart laboratory for helpful discussions. We thank Dr. B. Kallipolitis for providing the anti-Listeria Hfq antibody and Dr. E. Hajnsdorf for the anti-E.coli Hfq. We acknowledge the platforms of the Grenoble Instruct-ERIC Center (ISBG, UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 670823,H2020,ERC-2014-ADG,BacCellEpi(2015), Stavru, Fabrizia, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Bacterial, cellular and epigenetic factors that control enteropathogenicity - BacCellEpi - - H20202015-10-01 - 2018-09-30 - 670823 - VALID, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-10-INBS-05-01/10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR: 17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris, Laboratoire d’innovation : vaccins – Innovation lab : vaccines, Institut Pasteur [Paris], Biologie Evolutive de la Cellule Microbienne - Evolutionary Biology of the Microbial Cell, Génétique des Interactions Macromoléculaires, and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris
Subjects: [SDV]Life Sciences [q-bio], Virulence, RNA-binding protein, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, medicine.disease_cause, Microbiology, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Mice, 03 medical and health sciences, 0302 clinical medicine, Bacterial Proteins, Listeria monocytogenes, Virology, Extracellular, medicine, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Animals, Humans, Pathogen, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, 030304 developmental biology, 0303 health sciences, Innate immune system, Host Microbial Interactions, RIG-I, RNA-Binding Proteins, food and beverages, Interferon-beta, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Immunity, Innate, extracellular RNA, 3. Good health, Cell biology, RNA, Bacterial, HEK293 Cells, DEAD Box Protein 58, Parasitology, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030217 neurology & neurosurgery, bacteriophage A118, type I IFN, Signal Transduction, Extracellular RNA
Abstract: International audience; RNA-binding proteins (RBPs) perform key cellular activities by controlling the function of bound RNAs. The widely held assumption that RBPs are strictly intracellular has been challenged by the discovery of secreted RBPs. However, extracellular RBPs have been described in eukaryotes, while secreted bacterial RBPs have not been reported. Here, we show that the bacterial pathogen Listeria monocytogenes secretes a small RBP that we named Zea. We show that Zea binds a subset of L. monocytogenes RNAs, causing their accumulation in the extracellular medium. Furthermore, during L. monocytogenes infection, Zea binds RIG-I, the non-self-RNA innate immunity sensor, potentiating interferon-β production. Mouse infection studies reveal that Zea affects L. monocytogenes virulence. Together, our results unveil that bacterial RNAs can be present extracellularly in association with RBPs, acting as “social RNAs” to trigger a host response during infection.
Published: 2019
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30. Complex Formation between Mur Enzymes from Streptococcus pneumoniae

Author: Christine Ebel, Adriana Franco Paes Leme, Andréa Dessen, Daniel M. Trindade, Daniela C. Granato, Mayara M. Miyachiro, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0303 health sciences, Multiprotein complex, Lipid II, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Microscale thermophoresis, 030302 biochemistry & molecular biology, Biochemistry, Bacterial cell structure, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, chemistry, Cytoplasm, Inner membrane, Peptidoglycan
Abstract: International audience; Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen Streptococcus pneumoniae and characterized their interactions using chemical cross-linking, mass spectrometry, analytical ultracentrifugation, and microscale thermophoresis. Mur ligases interact strongly as binary complexes, with interaction regions mapping mostly to loop regions. Interestingly, MurC, MurD, and MurE display 10-fold higher affinity for each other than for MurF and MurG, suggesting that Mur ligases that catalyze the initial reactions in the peptidoglycan biosynthesis pathway could form a subcomplex that could be important to facilitate Lipid II biosynthesis. The interface between Mur proteins could represent a yet unexplored target for new inhibitor studies that could lead to the development of novel antimicrobials.
Published: 2019
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31. Complex Formation between Mur Enzymes from

Author: Mayara M, Miyachiro, Daniela, Granato, Daniel Maragno, Trindade, Christine, Ebel, Adriana Franco, Paes Leme, and Andréa, Dessen
Subjects: Streptococcus pneumoniae, Bacterial Proteins, Humans, Amino Acid Sequence, Protein Structure, Secondary, Protein Binding
Abstract: Peptidoglycan is one of the major components of the bacterial cell wall, being responsible for shape and stability. Due to its essential nature, its biosynthetic pathway is the target for major antibiotics, and proteins involved in its biosynthesis continue to be targeted for inhibitor studies. The biosynthesis of its major building block, Lipid II, is initiated in the bacterial cytoplasm with the sequential reactions catalyzed by Mur enzymes, which have been suggested to form a multiprotein complex to facilitate shuttling of the building blocks toward the inner membrane. In this work, we purified MurC, MurD, MurE, MurF, and MurG from the human pathogen
Published: 2019

32. An RNA-binding protein secreted byListeria monocytogenesactivates RIG-I signaling

Author: Mikael Koutero, Valérie Najburg, Christophe Bécavin, Bruno Dupuy, To Nam Tham, Alessandro Pagliuso, Stevens Robertin, Pascale Cossart, Fabrizia Stavru, Alice Lebreton, Eric Allemand, Quentin Bertrand, Marie-Anne Nahori, Anastassia V. Komarova, Christian Muchard, and Andréa Dessen
Subjects: 0303 health sciences, Innate immune system, 030306 microbiology, RIG-I, fungi, food and beverages, RNA-binding protein, Biology, medicine.disease_cause, 3. Good health, Cell biology, 03 medical and health sciences, Crosstalk (biology), Listeria monocytogenes, Extracellular, medicine, Extracellular ribonucleoprotein complex, Intracellular, 030304 developmental biology
Abstract: SummaryRecent studies have reported on the presence of bacterial RNA within or outside extracellular membrane vesicles, possibly as ribonucleoprotein complexes. Proteins that bind and stabilize bacterial RNAs in the extracellular environment have not been reported. Here, we show that the bacterial pathogenListeria monocytogenessecretes a small RNA binding protein that we named Zea. We show that Zea binds and stabilizes a subset ofL. monocytogenesRNAs causing their accumulation in the extracellular medium. Furthermore, Zea binds RIG-I, the vertebrate non-self-RNA innate immunity sensor and potentiates RIG-I-signaling leading to interferon β production. By performingin vivoinfection, we finally show that Zea modulatesL. monocytogenesvirulence. Together, this study reveals that bacterial extracellular RNAs and RNA binding proteins can affect the host-pathogen crosstalk.
Published: 2019
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33. Penicillin-Binding Proteins (PBPs) and Bacterial Cell Wall Elongation Complexes

Author: Andréa Dessen, Mayara M. Miyachiro, Carlos Contreras-Martel, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and ANR-18-CE11-0019,PSEUDO-WALL,Architecture et fonction de complexes de la biosynthèse de la paroi cellulaire de Pseudomonas aeruginosa(2018)
Subjects: Penicillin binding proteins, Protein complexes, Peptidoglycan, Bacterial cell structure, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, MESH: Cell Wall, Membrane proteins, medicine, Penicillin-Binding proteins (PBPs), Cell wall elongation, 030304 developmental biology, 0303 health sciences, MESH: Penicillin-Binding Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, MESH: Peptidoglycan, Periplasmic space, Cell biology, Penicillin, MESH: Bacteria, chemistry, Membrane protein, Structural biology, Periplasm, medicine.drug
Abstract: International audience; The bacterial cell wall is the validated target of mainstream antimicrobials such as penicillin and vancomycin. Penicillin and other β-lactams act by targeting Penicillin-Binding Proteins (PBPs), enzymes that play key roles in the biosynthesis of the main component of the cell wall, the peptidoglycan. Despite the spread of resistance towards these drugs, the bacterial cell wall continues to be a major Achilles' heel for microbial survival, and the exploration of the cell wall formation machinery is a vast field of work that can lead to the development of novel exciting therapies. The sheer complexity of the cell wall formation process, however, has created a significant challenge for the study of the macromolecular interactions that regulate peptidoglycan biosynthesis. New developments in genetic and biochemical screens, as well as different aspects of structural biology, have shed new light on the importance of complexes formed by PBPs, notably within the cell wall elongation machinery. This chapter summarizes structural and functional details of PBP complexes involved in the periplasmic and membrane steps of peptidoglycan biosynthesis with a focus on cell wall elongation. These assemblies could represent interesting new targets for the eventual development of original antibacterials.
Published: 2019
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34. Pore-forming activity of the $Pseudomonas\ aeruginosa$ type III secretion system translocon alters the host epigenome

Author: Charlotte Lombardi, Alain Filloux, Laurent Dortet, François Cretin, Andréa Dessen, AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), MRC Centre for Molecular Microbiology and Infection, Department of Life Sciences, Imperial College London, London, United Kingdom, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse bactérienne et réponses cellulaires (PBRC), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Vaincre la Mucoviscidose RF20140501133, ANR project PRP1.4 T3SS, European Project: 654909,H2020,H2020-MSCA-IF-2014,T6SS-PSEUDO-LIP(2016), Université Grenoble Alpes, Inserm, CEA, BIG-Biologie du Cancer et de l’Infection, Grenoble, France, Imperial College London, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Medical Research Council (MRC)
Subjects: 0301 basic medicine, EFFECTOR TARGETS, NF-KAPPA-B, Moths, medicine.disease_cause, Applied Microbiology and Biotechnology, Virulence factor, Epigenesis, Genetic, Type three secretion system, Histones, INFECTION, Type III Secretion Systems, MESH: Animals, MESH: Epigenesis, Genetic, PHOSPHORYLATION, MESH: Bacterial Proteins, Host cell membrane, MESH: Histones, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Effector, Chemistry, MESH: Moths, EPITHELIAL-CELLS, Translocon, Cell biology, Histone Code, Larva, Host-Pathogen Interactions, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, Life Sciences & Biomedicine, Microbiology (medical), GENES, Immunology, Microbiology, 03 medical and health sciences, MESH: Type III Secretion Systems, Bacterial Proteins, Genetics, medicine, Animals, Humans, Secretion, HISTONE H3, PROTEIN PHOSPHATASES, Antigens, Bacterial, Science & Technology, MESH: Humans, Cell Membrane, MESH: Host-Pathogen Interactions, Cell Biology, Epigenome, biochemical phenomena, metabolism, and nutrition, MAPK, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, MESH: Histone Code, A549 Cells, MESH: HeLa Cells, bacteria, MESH: A549 Cells, VI SECRETION, MESH: Larva, MESH: Antigens, Bacterial, HeLa Cells, MESH: Cell Membrane
Abstract: Recent studies highlight that bacterial pathogens can reprogram target cells by influencing epigenetic factors. The type III secretion system (T3SS) is a bacterial nanomachine that resembles a syringe on the bacterial surface. The T3SS ‘needle’ delivers translocon proteins into eukaryotic cell membranes, subsequently allowing injection of bacterial effectors into the cytosol. Here we show that Pseudomonas aeruginosa induces early T3SS-dependent dephosphorylation and deacetylation of histone H3 in eukaryotic cells. This is not triggered by any of the P. aeruginosa T3SS effectors, but results from the insertion of the PopB–PopD translocon into the membrane. This suggests that the P. aeruginosa translocon is a genuine T3SS effector acting as a pore-forming toxin. We visualized the translocon plugged into the host cell membrane after the bacterium has left the site of contact, and demonstrate that subsequent ion exchange through this pore is responsible for histone H3 modifications and host cell subversion. The pore-forming activity of the Pseudomonas aeruginosa type III secretion system translocon serves as a virulence factor to induce host epigenome modification and promote infection.
Published: 2018
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35. Assembly of an atypical α-macroglobulin complex from Pseudomonas aeruginosa

Author: Cécile Breyton, Christine Ebel, Marko Nedeljković, Andréa Dessen, Viviana Job, Ina Attrée, Daniel M. Trindade, Mylène Robert-Genthon, Samira Zouhir, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0301 basic medicine, Proteases, Operon, medicine.medical_treatment, 030106 microbiology, lcsh:Medicine, medicine.disease_cause, Thioester, Article, Microbiology, 03 medical and health sciences, Immune system, Bacterial Proteins, medicine, lcsh:Science, chemistry.chemical_classification, Multidisciplinary, Protease, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Pseudomonas aeruginosa, lcsh:R, Periplasmic space, biology.organism_classification, Pregnancy-Associated alpha 2-Macroglobulins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, chemistry, lcsh:Q, Protein Multimerization, Bacteria
Abstract: Alpha-2-macroglobulins (A2Ms) are large spectrum protease inhibitors that are major components of the eukaryotic immune system. Pathogenic and colonizing bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, also carry structural homologs of eukaryotic A2Ms. Two types of bacterial A2Ms have been identified: Type I, much like the eukaryotic form, displays a conserved thioester that is essential for protease targeting, and Type II, which lacks the thioester and to date has been poorly studied despite its ubiquitous presence in Gram-negatives. Here we show that MagD, the Type II A2M from P. aeruginosa that is expressed within the six-gene mag operon, specifically traps a target protease despite the absence of the thioester motif, comforting its role in protease inhibition. In addition, analytical ultracentrifugation and small angle scattering show that MagD forms higher order complexes with proteins expressed in the same operon (MagA, MagB, and MagF), with MagB playing the key stabilization role. A P. aeruginosa strain lacking magB cannot stably maintain MagD in the bacterial periplasm, engendering complex disruption. This suggests a regulated mechanism of Mag complex formation and stabilization that is potentially common to numerous Gram-negative organisms, and that plays a role in periplasm protection from proteases during infection or colonization.
Published: 2018
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36. Identificação de novos inibidores de PBPs, agentes microbianos em potencial

Author: Paulo Vinicius da Mata Madeira, Caíque C. Malospirito, and Andréa Dessen
Published: 2017
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37. New strategies for targeting and treatment of multi-drug resistant Staphylococcus aureus

Author: Marko Nedeljković, Andréa Dessen, L. Mayrink Assis, Brazilian National Laboratory for Biosciences (LNBio), Centro Nacional de Pesquisa em Energia e materiais, Centra Nacional de Pesquisa-Centra Nacional de Pesquisa, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: 0301 basic medicine, Drug, Staphylococcus aureus, Cancer Research, medicine.drug_class, media_common.quotation_subject, 030106 microbiology, Antibiotics, Virulence, Biology, medicine.disease_cause, Microbiology, 03 medical and health sciences, Antibiotic resistance, Cell Wall, Drug Resistance, Multiple, Bacterial, Drug Discovery, medicine, Animals, Humans, Penicillin-Binding Proteins, Pharmacology (medical), ComputingMilieux_MISCELLANEOUS, media_common, Pharmacology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Staphylococcal Infections, Anti-Bacterial Agents, 3. Good health, Community-Acquired Infections, 030104 developmental biology, Infectious Diseases, Oncology, Mutation, Multi drug resistant, Efflux, Staphylococcus
Abstract: Staphylococcus aureus is a major cause of bacterial infection in humans, and has been notoriously able to acquire resistance to a variety of antibiotics. An example is methicillin-resistant S. aureus (MRSA), which despite having been initially associated with clinical settings, now is one of the key causative agents of community-acquired infections. Antibiotic resistance in S. aureus involves mechanisms ranging from drug efflux to increased expression or mutation of target proteins, and this has required innovative approaches to develop novel treatment methodologies. This review provides an overview of the major mechanisms of antibiotic resistance developed by S. aureus, and describes the emerging alternatives being sought to circumvent infection and proliferation, including new generations of classic antibiotics, synergistic approaches, antibodies, and targeting of virulence factors.
Published: 2017
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38. Structural Similarity of Secretins from Type II and Type III Secretion Systems

Author: Anthony P. Pugsley, Andréa Dessen, Ingrid Guilvout, Tommaso Tosi, Viviana Job, Guy Schoehn, Leandro F. Estrozi, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Génétique Moléculaire, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Biologie structurale des interactions entre virus et cellule hôte (UVHCI), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS), Brazilian National Laboratory for Biosciences (LNBio), Centro Nacional de Pesquisa em Energia e materiais, Centra Nacional de Pesquisa-Centra Nacional de Pesquisa, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, MESH: Protein Structure, Quaternary, Structural similarity, [SDV]Life Sciences [q-bio], MESH: Klebsiella oxytoca, Biology, digestive system, Type three secretion system, Microbiology, Saucer, 03 medical and health sciences, fluids and secretions, Secretin, Structural Biology, Secretion, MESH: Bacterial Secretion Systems, Protein Structure, Quaternary, Bacterial Secretion Systems, Molecular Biology, MESH: Structural Homology, Protein, 030304 developmental biology, MESH: Secretin, 0303 health sciences, Type II secretion system, MESH: Bacterial Outer Membrane Proteins, Cryoelectron Microscopy, 030302 biochemistry & molecular biology, Klebsiella oxytoca, Secretory protein, Structural Homology, Protein, Bacterial Outer Membrane Proteins, MESH: Pseudomonas aeruginosa, Pseudomonas aeruginosa, Biophysics, MESH: Cryoelectron Microscopy, Bacterial outer membrane, MESH: Models, Molecular, hormones, hormone substitutes, and hormone antagonists
Abstract: International audience; 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.
Published: 2014
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39. Structural Basis of Pilus Anchoring by the Ancillary Pilin RrgC of Streptococcus pneumoniae

Author: Amandine Maccagni, Munan Shaik, Andréa Dessen, Anne Marie Di Guilmi, Guillaume Tourcier, Thomas, Frank, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Sequence Data, MESH: Amino Acid Sequence, Plasma protein binding, medicine.disease_cause, Biochemistry, Pilus, Bacterial cell structure, MESH: Fimbriae, Bacterial, Microbiology, Fimbriae Proteins, MESH: Protein Structure, Tertiary, chemistry.chemical_compound, Bacterial Proteins, MESH: Aminoacyltransferases, Streptococcus pneumoniae, medicine, MESH: Protein Binding, Amino Acid Sequence, MESH: Bacterial Proteins, Molecular Biology, MESH: Molecular Sequence Data, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, Cell Biology, biochemical phenomena, metabolism, and nutrition, Aminoacyltransferases, MESH: Fimbriae Proteins, Protein Structure, Tertiary, 3. Good health, Bacterial adhesin, Cysteine Endopeptidases, chemistry, Fimbriae, Bacterial, Pilin, Protein Structure and Folding, biology.protein, bacteria, Peptidoglycan, MESH: Streptococcus pneumoniae, Protein Binding, MESH: Cysteine Endopeptidases
Abstract: International audience; 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.
Published: 2014
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40. Resistance to antibiotics targeted to the bacterial cell wall

Author: Andréa Dessen, I Nikolaidis, and S Favini-Stabile
Subjects: 0303 health sciences, Penicillin binding proteins, Lipid II, 030306 microbiology, Lysin, Periplasmic space, Biology, Biochemistry, Bacterial cell structure, 03 medical and health sciences, chemistry.chemical_compound, chemistry, Cytoplasm, Peptidoglycan, Molecular Biology, Cellular compartment, 030304 developmental biology
Abstract: Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional 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.
Published: 2014
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41. Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Author: Adrian J. Lloyd, Dominique Mengin-Lecreulx, Andréa Dessen, Karen M. Ruane, Hélène Barreteau, Didier Blanot, David I. Roper, Sébastien Dementin, Vilmos Fülöp, Stanislav Gobec, Christopher G. Dowson, Audrey Boniface, From the School of Life Sciences, University of Warwick [Coventry], Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: Lysine, Bacterial Metabolism, Crystallography, X-Ray, Cell morphology, Biochemistry, Pentapeptide repeat, MESH: Protein Structure, Tertiary, chemistry.chemical_compound, X-ray Crystallography, Structural Biology, Cell Wall, MESH: Staphylococcus aureus, Peptide Synthases, MESH: Metabolomics, MESH: Bacterial Proteins, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Peptidoglycan, MESH: Peptide Synthases, Amino acid, Amino Acid, Staphylococcus aureus, Peptidoglycan, Biology, Microbiology, complex mixtures, Cell wall, QH301, 03 medical and health sciences, MurE, MESH: Cell Wall, Bacterial Proteins, Consensus sequence, Metabolomics, MESH: Lysine, Molecular Biology, 030304 developmental biology, Enzyme Kinetics, DNA ligase, 030306 microbiology, Cell Biology, MESH: Crystallography, X-Ray, Protein Structure, Tertiary, QR, chemistry, Antibiotic Resistance, bacteria
Abstract: Background: MurE controls stereochemical incorporation of lysine or diaminopimelate into peptidoglycan stem peptides. Results: The structure of S. aureus MurE reveals an unexpected lack of specificity for lysine within the active site. Conclusion: Incorporation of lysine is supported by the comparatively high concentration of cytoplasmic lysine, not enzyme specificity. Significance: This study provides new perspectives in targeting Gram-positive peptidoglycan assembly for antimicrobial discovery., Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.
Published: 2013
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42. Structure of Internalin InlK from the Human Pathogen Listeria monocytogenes

Author: David Neves, Andréa Dessen, Laurent Dortet, Viviana Job, Pascale Cossart, Brazilian National Laboratory for Biosciences (LNBio), Centro Nacional de Pesquisa em Energia e materiais, Centra Nacional de Pesquisa-Centra Nacional de Pesquisa, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [11/52067-6], Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, autophagy, Protein Conformation, Virulence Factors, Molecular Sequence Data, MESH: Sequence Alignment, Virulence, Human pathogen, MESH: Amino Acid Sequence, Leucine-rich repeat, MESH: Listeria monocytogenes, medicine.disease_cause, virulence factor, Virulence factor, Microbiology, protein-protein interaction, 03 medical and health sciences, MESH: Protein Conformation, Bacterial Proteins, Listeria monocytogenes, Structural Biology, Major vault protein, medicine, Humans, Protein Interaction Domains and Motifs, Internalin, Amino Acid Sequence, MESH: Bacterial Proteins, Molecular Biology, X-ray crystallography, MESH: Virulence Factors, 030304 developmental biology, MESH: Protein Interaction Domains and Motifs, 0303 health sciences, MESH: Humans, MESH: Molecular Sequence Data, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, 030302 biochemistry & molecular biology, bacterial infection, biology.organism_classification, Listeria, biology.protein, Sequence Alignment, MESH: Models, Molecular
Abstract: International audience; 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.
Published: 2013
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43. Structure–activity relationships of new cyanothiophene inhibitors of the essential peptidoglycan biosynthesis enzyme MurF

Author: Stanislav Gobec, Samo Turk, Hélène Barreteau, Dominique Mengin-Lecreulx, Andréa Dessen, Alex J. O'Neill, Didier Blanot, Christopher P. Randall, Izidor Sosič, Carlos Contreras-Martel, Martina Hrast, Damijan Knez, Faculty of Pharmacy, University of Ljubljana, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, MESH: Catalytic Domain, Microbial Sensitivity Tests, Peptidoglycan, Thiophenes, MESH: Drug Design, medicine.disease_cause, 01 natural sciences, Bacterial cell structure, Structure-Activity Relationship, 03 medical and health sciences, chemistry.chemical_compound, MESH: Structure-Activity Relationship, Biosynthesis, Catalytic Domain, MESH: Anti-Bacterial Agents, Drug Discovery, medicine, Nucleotide, Enzyme Inhibitors, Peptide Synthases, Escherichia coli, 030304 developmental biology, Pharmacology, chemistry.chemical_classification, MESH: Microbial Sensitivity Tests, 0303 health sciences, DNA ligase, Bacteria, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Peptidoglycan, 010405 organic chemistry, Organic Chemistry, General Medicine, MESH: Thiophenes, MESH: Peptide Synthases, Anti-Bacterial Agents, 0104 chemical sciences, MESH: Bacteria, Enzyme, chemistry, Biochemistry, MESH: Enzyme Inhibitors, Drug Design, Antibacterial activity, MESH: Models, Molecular
Abstract: International audience; Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials.
Published: 2013
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44. MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis

Author: Nicole M. Thielens, Sandy Favini-Stabile, Andréa Dessen, and Carlos Contreras-Martel
Subjects: chemistry.chemical_classification, 0303 health sciences, DNA ligase, biology, 030306 microbiology, Periplasmic space, biology.organism_classification, Microbiology, MreB, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, chemistry, Biochemistry, Cytoplasm, Thermotoga maritima, Peptidoglycan, Cytoskeleton, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology
Abstract: 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.
Published: 2013
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45. Structural basis of eukaryotic cell targeting by type III secretion system (T3SS) effectors

Author: David Neves, Alexander Pflug, Andréa Dessen, Karen F. Discola, Tommaso Tosi, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Hôpital Henri Mondor, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Henri Mondor-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Thomas, Frank
Subjects: MESH: Protein Transport, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Peptide Hydrolases, Biology, MESH: Eukaryotic Cells, Microbiology, Type three secretion system, MESH: Secretory Pathway, 03 medical and health sciences, Animals, Humans, MESH: Animals, MESH: Proteins, Secretion, Molecular Biology, Secretory pathway, 030304 developmental biology, 0303 health sciences, Secretory Pathway, MESH: Humans, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 030306 microbiology, Effector, Proteins, General Medicine, Cell biology, Transport protein, Protein Transport, Eukaryotic Cells, Structural biology, Cytoplasm, Function (biology), Peptide Hydrolases
Abstract: International audience; 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.
Published: 2013
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46. Structural and mechanistic basis of penicillin-binding protein inhibition by lactivicins

Author: Tom Brown, André Luxen, Pauline Macheboeuf, Christopher J. Schofield, Andréa Dessen, Astrid Zervosen, Delphine S Fischer, and Bernard Joris
Subjects: Cefotaxime, Penicillin binding proteins, medicine.drug_class, Antibiotics, Microbial Sensitivity Tests, medicine.disease_cause, Crystallography, X-Ray, Peptides, Cyclic, Bacterial cell structure, Lactivicin, Drug Resistance, Bacterial, medicine, polycyclic compounds, Penicillin-Binding Proteins, Molecular Biology, biology, Chemistry, Cycloserine, Pathogenic bacteria, Cell Biology, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, Streptococcus pneumoniae, Biochemistry, Peptides, Bacteria, medicine.drug, Protein Binding
Abstract: Beta-lactam antibiotics, including penicillins and cephalosporins, inhibit penicillin-binding proteins (PBPs), which are essential for bacterial cell wall biogenesis. Pathogenic bacteria have evolved efficient antibiotic resistance mechanisms that, in Gram-positive bacteria, include mutations to PBPs that enable them to avoid beta-lactam inhibition. Lactivicin (LTV; 1) contains separate cycloserine and gamma-lactone rings and is the only known natural PBP inhibitor that does not contain a beta-lactam. Here we show that LTV and a more potent analog, phenoxyacetyl-LTV (PLTV; 2), are active against clinically isolated, penicillin-resistant Streptococcus pneumoniae strains. Crystallographic analyses of S. pneumoniae PBP1b reveal that LTV and PLTV inhibition involves opening of both monocyclic cycloserine and gamma-lactone rings. In PBP1b complexes, the ring-derived atoms from LTV and PLTV show a notable structural convergence with those derived from a complexed cephalosporin (cefotaxime; 3). The structures imply that derivatives of LTV will be useful in the search for new antibiotics with activity against beta-lactam-resistant bacteria.
Published: 2016
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47. Structural Basis of Lipid Targeting and Destruction by the Type V Secretion System of Pseudomonas aeruginosa

Author: Pauline Basso, Sophie Bleves, Carlos Contreras-Martel, Eric Faudry, David Neves, Andréa Dessen, Aurélie Laubier, Richard Salacha, Paulo Vinicius da Mata Madeira, Samira Zouhir, Brazilian National Laboratory for Biosciences (LNBio), Centro Nacional de Pesquisa em Energia e materiais, Centra Nacional de Pesquisa-Centra Nacional de Pesquisa, Pathogenèse bactérienne et réponses cellulaires (PBRC), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Laboratoire International Associé BACWALL, Association Vaincre la Mucoviscidose - RF20120600685 et RF20130500911, FAPESP - 11/52067-6, 2014/11980-9 et 2013/01962-0, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: Models, Molecular, 0301 basic medicine, Protein Conformation, Type V Secretion Systems, 030106 microbiology, Virulence, Phospholipase, Biology, Crystallography, X-Ray, Phosphatidylinositols, Substrate Specificity, 03 medical and health sciences, Bacterial Proteins, Phospholipase A1, Structural Biology, Catalytic Domain, Hydrolase, Secretion, Lipase, Molecular Biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Active site, Transport protein, 030104 developmental biology, Biochemistry, Phospholipases, Pseudomonas aeruginosa, biology.protein
Abstract: International audience; 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.
Published: 2016
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48. Structural Insights into Protein-Protein Interactions Involved in Bacterial Cell Wall Biogenesis

Author: Mayara M. Miyachiro, Andréa Dessen, Federica Laddomada, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Brazilian National Laboratory for Biosciences (LNBio), Centro Nacional de Pesquisa em Energia e materiais, Centra Nacional de Pesquisa-Centra Nacional de Pesquisa, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: cell division, 0301 basic medicine, Microbiology (medical), Cell division, 030106 microbiology, elongation, Review, peptidoglycan, Biology, Biochemistry, Microbiology, bacterial cytoskeleton, Protein–protein interaction, MraY, Prokaryotic cytoskeleton, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Pharmacology (medical), General Pharmacology, Toxicology and Pharmaceutics, Cytoskeleton, protein complexes, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], lcsh:RM1-950, Cell biology, lcsh:Therapeutics. Pharmacology, 030104 developmental biology, Infectious Diseases, chemistry, Cytoplasm, Peptidoglycan, Mur enzymes, Biogenesis
Abstract: International audience; 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.
Published: 2016
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49. Calcium-Dependent Complex Formation Between PBP2 and Lytic Transglycosylase SltB1 ofPseudomonas aeruginosa

Author: Eefjan Breukink, Ioulia Nikolaidis, Nicole M. Thielens, Andréa Dessen, Viviana Job, and Thierry Izoré
Subjects: Models, Molecular, Microbiology (medical), Penicillin binding proteins, Cell division, Molecular Sequence Data, Immunology, Peptidoglycan, Biology, Crystallography, X-Ray, Microbiology, Bacterial cell structure, Cell wall, chemistry.chemical_compound, Bacterial Proteins, Cell Wall, Escherichia coli, Penicillin-Binding Proteins, Protein Interaction Domains and Motifs, Amino Acid Sequence, Cloning, Molecular, Pharmacology, Binding Sites, Glycosyltransferases, Periplasmic space, Recombinant Proteins, chemistry, Lytic cycle, Biochemistry, Cytoplasm, Pseudomonas aeruginosa, Calcium, Sequence Alignment, Protein Binding
Abstract: In Gram-negative bacteria, the bacterial cell wall biosynthetic mechanism requires the coordinated action of enzymes and structural proteins located in the cytoplasm, within the membrane, and in the periplasm of the cell. Its main component, peptidoglycan (PG), is essential for cell division and wall elongation. Penicillin-binding proteins (PBPs) catalyze the last steps of PG biosynthesis, namely the polymerization of glycan chains and the cross-linking of stem peptides, and can be either monofunctional or bifunctional. Their action is coordinated with that of other enzymes essential for cell-wall biosynthesis, such as lytic transglycosylases (LT). Here, we have studied SltB1, an LT from Pseudomonas aeruginosa, and identified that it forms a complex with PBP2, a monofunctional enzyme, which requires the presence of Ca(2+). In addition, we have solved the structure of SltB1 to a high resolution, and identified that it harbors an EF-hand like motif containing a Ca(2+) ion displaying bipyramidal coordination. These studies provide initial structural details that shed light on the interactions between the PG biosynthesis enzymes in P. aeruginosa.
Published: 2012
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50. Characterization of the elongasome core PBP2 : MreC complex of Helicobacter pylori

Author: Sylviane Hoos, Christine Ebel, Alexandre Martins, Christine Schmitt, Frank Gabel, Chantal Ecobichon, Patrick England, Andréa Dessen, Pierre-Jean Matteï, Ivo G. Boneca, Meriem El Ghachi, Marie-Christine Prévost, and Frédéric Colland
Subjects: chemistry.chemical_classification, 0303 health sciences, 030306 microbiology, Cell, Periplasmic space, biochemical phenomena, metabolism, and nutrition, Biology, Cell morphology, Microbiology, Yeast, Bacterial cell structure, Amino acid, law.invention, 03 medical and health sciences, medicine.anatomical_structure, chemistry, Biochemistry, law, polycyclic compounds, Recombinant DNA, Biophysics, medicine, Surface plasmon resonance, Molecular Biology, 030304 developmental biology
Abstract: Summary The definition of bacterial cell shape is a complex process requiring the participation of multiple components of an intricate macromolecular machinery. We aimed at characterizing the determinants involved in cell shape of the helical bacterium Helicobacter pylori. Using a yeast two-hybrid screen with the key cell elongation protein PBP2 as bait, we identified an interaction between PBP2 and MreC. The minimal region of MreC required for this interaction ranges from amino acids 116 to 226. Using recombinant proteins, we showed by affinity and size exclusion chromatographies and surface plasmon resonance that PBP2 and MreC form a stable complex. In vivo, the two proteins display a similar spatial localization and their complex has an apparent 1:1 stoichiometry; these results were confirmed in vitro by analytical ultracentrifugation and chemical cross-linking. Small angle X-ray scattering analyses of the PBP2 : MreC complex suggest that MreC interacts directly with the C-terminal region of PBP2. Depletion of either PBP2 or MreC leads to transition into spherical cells that lose viability. Finally, the specific expression in trans of the minimal interacting domain of MreC with PBP2 in the periplasmic space leads to cell rounding, suggesting that the PBP2/MreC complex formation in vivo is essential for cell morphology.
Published: 2011
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