A New Catalytic Dyad Regulates Anchoring of Molecules to the Gram-Positive Cell Wall by Sortases

AbstractIn this issue of Structure, three crystal structures of staphylococcal sortase B reveal a new active site for cysteine proteases, which requires a Cys-Arg catalytic dyad for substrate turnover

Conformational States of a Bacterial α2-Macroglobulin Resemble Those of Human Complement C3

α2 macroglobulins (α2Ms) 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, α2Ms 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, α2M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α2M 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

Type III secretion proteins PcrV and PcrG from Pseudomonas aeruginosa form a 1:1 complex through high affinity interactions

BACKGROUND: Pseudomonas aeruginosa, an increasingly prevalent opportunistic pathogen, utilizes a type III secretion system for injection of toxins into host cells in order to initiate infection. A crucial component of this system is PcrV, which is essential for cytotoxicity and is found both within the bacterial cytoplasm and localized extracellularly, suggesting that it may play more than one role in Pseudomonas infectivity. LcrV, the homolog of PcrV in Yersinia, has been proposed to participate in effector secretion regulation by interacting with LcrG, which may act as a secretion blocker. Although PcrV also recognizes PcrG within the bacterial cytoplasm, the roles played by the two proteins in type III secretion in Pseudomonas may be different from the ones suggested for their Yersinia counterparts. RESULTS: In this work, we demonstrate by native mass spectrometry that PcrV and PcrG expressed and purified from E. coli form a 1:1 complex in vitro. Circular dichroism results indicate that PcrG is highly unstable in the absence of PcrV; in contrast, both PcrV alone and the PcrV:PcrG complex have high structural integrity. Surface plasmon resonance measurements show that PcrV interacts with PcrG with nanomolar affinity (15.6 nM) and rapid kinetics, an observation which is valid both for the full-length form of PcrG (residues 1–98) as well as a form which lacks the C-terminal 24 residues, which are predicted to have low secondary structure content. CONCLUSIONS: PcrV is a crucial component of the type III secretion system of Pseudomonas, but the way in which it participates in toxin secretion is not understood. Here we have characterized the interaction between PcrV and PcrG in vitro, and shown that PcrG is highly unstable. However, it associates readily with PcrV through a region located within its first 74 amino acids to form a high affinity complex. The fact that PcrV associates and dissociates quickly from an unstable molecule points to the transient nature of a PcrV:PcrG complex. These results are in agreement with analyses from pcrV deletion mutants which suggest that PcrV:PcrG may play a different role in effector secretion than the one described for the LcrV:LcrG complex in Yersinia

A bacterial protein targets the BAHD1 chromatin complex to stimulate type III interferon response

International audienceIntracellular pathogens such as $Listeria\ monocytogenes$ subvert cellular functions through the interaction of bacterial effectors with host components. Here we found that a secreted listerial virulence factor, LntA, could target the chromatin repressor BAHD1 in the host cell nucleus to activate interferon IFN-stimulated genes (ISGs). IFN-$\lambda$ expression was induced in response to infection of epithelial cells with bacteria lacking LntA; however, the BAHD1-chromatin associated complex repressed downstream ISGs. In contrast, in cells infected with $lntA$-expressing bacteria, LntA prevented BAHD1 recruitment to ISGs and stimulated their expression. Murine listeriosis decreased in BAHD1$^{+/-}$ mice or when $lntA$ was constitutively expressed. Thus, the LntA-BAHD1 interplay may modulate IFN-$\lambda$-mediated immune response to control bacterial colonization of the host

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

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

Structural Basis of Cytotoxicity Mediated by the Type III Secretion Toxin ExoU from Pseudomonas aeruginosa

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

Hijacking of the Pleiotropic Cytokine Interferon-γ by the Type III Secretion System of Yersinia pestis

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

Caractérisation structurale et fonctionnelle des composants du pilus de Streptococcus pneumoniae (vers une meilleure compréhension de la biogenèse des pili)

Streptococcus pneumoniae est un pathogène majeur chez l'homme, responsable d'otites sévères, de pneumonies, de bactériémies et de méningites. C'est une cause majeure de mortalité et de morbidité, essentiellement chez les enfants et les personnes âgées. Il a été récemment découvert que certaines souches virulentes de S. pneumoniae portent à leur surface des pili, considérés comme un important facteur de virulence. Cependant, contrairement aux bactéries à Gram-négatif, peu de choses sont connues sur la structure des pili des bactéries à Gram-positif. Les gènes requis pour la production des pili chez S. pneumoniae sont localisés sur un îlot de pathogénicité et codent pour 3 sortases (SrtC-l, SrtC-2, SrtC-3) et 3 protéines structurales (RrgB, qui forme le corps du pilus, RrgA et RrgC). Celles-ci contiennent un motif LPXTG, motif de reconnaissance des enzymes sortase. Dans ce travail, nous apportons de nouvelles informations structurale, biochimique et microbiologique sur le mécanisme de formation du pilus par (1) une reconstitution du corps de la fibre in vitro après avoir identifié SrtC-1 comme étant la principale pilus-polymérase; (2) une étude de microscopie électronique montrant que les fibres produites in vitro mimaient structuralement les pili ; (3) une étude in vivo confirmant les résultats obtenus in vitro; (4) la résolution de la structure par cristallographie aux rayons X de SrtC-l et SrtC-3 qui révèle un site actif dont l'accès est contrôlé par un couvercle flexible, contrairement aux sortases non impliquées dans la biogenèse de pili. Ces observations suggèrent que la spécificité de substrat est dictée par une reconnaissance de surface couplée à une ouverture du couvercle. Dans un second temps, nous avons caractérisé la région du site actif de SrtC-1 : la triade catalytique mais aussi les deux résidus du couvercle qui s'ancrent dans le site actif. L'identification de résidus clés dans l'activité sortasique aussi bien que dans la stabilisation structurale a été ainsi mis en évidence.Streptococcus pneumoniae, a piliated pathogen, is the causative agent of otitis, pneumonia, bacteremia, and meningitis. It is a major cause of community-acquired iIInesses, especially in the very young and the elderly. Pili have been shown to play key roles in pneumococcal infection, and although the pilus biogenesis mechanism has been weil studied in Gram-negative bacteria, it remains poorly understood in Gram-positive species. Pilus assembly in S. pneumoniae involves a membrane-associated macromolecular machinery encoded on a single pathogenicity islet, and includes the backbone fiber protein (RrgB), two minor pilins (RrgA and RrgC), and three dedicated sortases (SrtC-l, SrtC-2, SrtC-3). These pilin subunits contain the recognition LPXTG motif of sortases. Here, we provide structural, biochemical, and microbiological insight into this novel pilus formation mechanism by (1) reconstituting the main backbone fiber of the pneumococcal pilus in vitro upon identification that SrtC-1 acts as the main pilus-polymerase and can form RrgB fibers; (2) employing electron microscopy in order to visualize fibers produced in vitro and showing that they structurally mimic the pneumococcal pilus backbone; (3) confirming this result in vivo; (4) solving the high resolution crystal structures ofSrtC-1 and SrtC-3 which reveal active sites whose access is controlled by flexible lids, unlike in non- pilus sortases, suggesting that substrate specificity is dictated by surface recognition coupled to lid opening. ln addition, we characterize the catalytic triad present in the sortase active site as weil as lid residues, and identiJy key amino acids which are relevant for sortase activity as weil as structural stabilization.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Etudes structurale et fonctionnelle de protéines impliquées dans la virulence chez S. pneumoniae et P. aeruginosa

Cette thèse est composée de deux parties : Le première partie rend compte de l'étude structurale de la protéine RrgA. RrgA est associée au pilus du pathogène Streptococcus pneumoniae et participe aux premières étapes de colonisation chez l'hôte en se liant à plusieurs composés de la Matrice Extra Cellulaire. Nous avons résolu la structure de cette protéine à 1.9 Å par cristallographie aux rayons-X. RrgA posse de une structure allongée formée de quatre domaines alignés d'origine eucaryote et procaryote. En effet, trois domaines ayant des similarités structurales avec les IgG et le domaine Cna-B semblent servir de piédestal pour orienter et présenter le domaine fonctionnel de type Intégrine. Nous avons confirmé la formation de deux ponts isopeptidiques stabilisateurs par spectrométrie de masse. De plus, le domaine intégrine possède deux insertions particulières dont la présence pourrait être impliquée dans la reconnaissance des divers substrats par RrgA. La deuxième partie de cette thèse est axée sur l'étude structurale du complexe ATPase et de ExsB, la pilotine présumée du système de sécrétion de type III chez Pseudomonas aeruginosa, bactérie opportuniste et jouant un rôle majeur dans l'infection des patients atteints de mucoviscidose. Pour la première fois, nous avons mis au point un protocole d'expression et de purification sous forme soluble de l'ATPase PscN en complexe avec une protéine partenaire, PscL. Des cristaux de ce complexe ont été obtenus au robot du PSB. Par ailleurs, nous avons confirmé l'expression de la lipoprotéine ExsB chez P. aeruginosa que nous avons localisée au sein de la membrane externe. De plus, nous avons résolu la structure de cette protéine qui présente un nouveau repliement et qui établie les bases structurales pour l'étude des pilotines pour tous les systèmes de sécrétion de type III de la famille Ysc.This manuscript is made up of two parts The first part describes the structural study of RrgA from Streptococcus pneumoniae. This protein is a pilus-associated adhesin that is able to bind to several components of the Extra Cellular Matrix and thus, participates in the first steps of host colonization. We solved the structure of RrgA to 1.9 Å by X-Ray crystallography. We showed that RrgA folds into an elongated 4-domain structure, and these domains display both eukaryotic and prokaryotic origins. Actually, three out of the four domains are reminiscent of IgG and Cna-B structures and act like stalks to orient and display the large Integrin-like domain. We confirmed the presence of two isopeptide bonds by mass spectrometry and hypothesised that the two inserted arms in the integrin domain could explain the wide variety of substrates RrgA can bind. The second part of this manuscript focuses on the structural studies of the ATPase complex as well as ExsB, the putative pilotin of the type III secretion system from Pseudomonas aeruginosa. This bacterium is a major threat in hospital-acquired infections and the main pathogen found in cystic-fibrosis suffering patients. For the first time we were able to express and purify the ATPase PscN in complex with its partner PscL. Crystallization trials led to a very promising condition that is being refined. Moreover, we confirmed expression of the lipoprotein ExsB in P. aeruginosa that we localised in the outer membrane. To have a better understanding of this protein, we also solved its high-resolution structure that displays a novel fold and our study paves the way for coming studies concerning pilotins.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Etude structurale et fonctionnelle de protéines impliquées dans la pathogénie bactérienne

La première partie de cette thèse s'intéresse au complexe PBP2 :MreC impliqué dans l'élongation d' Helicobacter pylori, pathogène responsable de pathologies gastriques. Ce complexe a pu être mis en évidence in vitro dont nous avons déterminé la stoechiométrie par différentes méthodes. Ce complexe 1 : 1 a été analysé par diffusion des rayons X aux petits angles permettant de modéliser une enveloppe du complexe à basse résolution. Des cristaux du complexe ont été obtenus ainsi qu'un jeu de données à 2.7 A. Par remplacement moléculaire, un domaine très conservé des PBP a pu être placé mais la présence de MreC est encore discutable. La seconde partie s'intéresse au système de sécrétion de type III, facteur de virulence majeur chez Pseudomonas a eruginosa , bactérie causant des maladies nosocomiales. PopB et PopD, deux protéines membranaires, forment un pore au niveau de la membrane d'une cellule-cible, nécessaire à la translocation de toxines. PopB a été purifiée sous forme soluble en présence de sa chaperonne, PcrH, complexe dissociable en présence de détergents et lipides. PopB seule, serait capable d'oligomériser en hexamère en formant des structures annulaires. La topologie du pore a également été déterminée dans des liposomes montrant ainsi que Po pB est insérée dans la membrane alors que PopD n'est associée que superficiellement. Pour finir, la structure de PcrH, a été résolue avec un peptide de PopD. Les résidus de Pc rH intervenant dans ce complexe sont essentiels à la sécrétion de PopD et à la formation du pore de translocation. Nous cherchons actuellement de petites molécules qui seraient capables d'interagir avec Pc rH et empêcheraient son interaction avec PopB et PopD.The first part of this thesis focuses on the PBP2 :MreC complex which is implicated in the elogation steps of the cell wall of Helicobacter pylori, a pathogen involved in gastric adenocarcinoma. The existence of such a complex was demonstrated in vitro which we were able to determine its stoichiometry by different methods. This 1: 1 complex was also analyzed by Small Angle X-ray Scattering with which we could model an envelope at low resolution. Crystals were obtained which diffracted up to 2.7 A. By molecular replacement we were able to place a highly conserved domain of PBP but the presence of MreC is not yet certain. The second part focuses on the type III secretion system of Pseudomonas aeruginosa, major virulence factor of this bacterium. PopB and PopD, two membrane proteins, form a pore in the membrane of a host-cell, and are thus able to translocate toxins directly into the eukaryotic cytoplasm. We purified PopB in soluble form associated to its chaperone then dissociated it in the 'presence of detergents and lipids. PopB alone is then able to oligomerize into hexamers and form ring-like structures which are visible by electron microscopy. We then determined the topology of thE pore in liposomes. We have shown that PopB is embedded in the membrane whereas PopD is superficially associated to it. We have finally solved the structure of the chaperone of Po pB and PopD, PcrH, with a PopD synthetic peptide. The PcrH residues involved in this complex are essential for the secretion of PopD and thus formation of the translocation pore formation. We are currently searching for molecules that are able to interact with PcrH and thus prevent the interaction with PopB and PopD.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF