Mécanismes moléculaires de la biogenèse du pilus chez Streptococcus pneumoniae

2010-10-20Streptococcus pneumoniae is the most common cause of otitis, sinusitis, pneumonia, sepsis and meningitis. Recently, pili were identified at the surface of S. pneumoniae and were proposed to play a role in the initial step of host tissues colonisation. Six genes are involved in the pilus formation. Three of them encode structural proteins named pilins (RrgA, RrgB and RrgC), the three others encode specific enzymes called sortases, which catalyse the covalent association of the pilins (SrtC-1, SrtC-2 and SrtC-3). Pilus formation models have been proposed based on genetic studies, but no biochemical data explaining precisely the molecular process of pilus formation is yet available. The individual study of each pilin led to the identification of stabilising Lys-Asn intramolecular bonds in each protein. Moreover, the cristallographic structure of RrgA and RrgB provided precious informations regarding the adhesive properties as well as the mechanism of pilus assembly. Since the role of each sortase remains unclear, our aim was to elucidate, step by step, the molecular mechanisms involved in the pilus biogenesis. Therefore, we have developed a co-expression plateform allowing the production of the pilins subunits together with sortases in E.coli. This system allowed to decipher the substrate specificity of the sortases, to generate covalent pilin/pilin complexes, as well as pilin/sortase complexes and thus provided key information towards the understanding complex macromolecular process.Streptococcus pneumoniae est un pathogène majeur chez l'homme, responsable d'otites, de pneumonies, de septicémies et de méningites. Récemment des structures de type pilus ont été identifiées à la surface de S. pneumoniae et jouent un rôle important dans les étapes initiales de colonisation des tissus hôtes. Six gènes sont impliqués dans la formation de cette structure. Trois d'entre eux codent pour les protéines structurales ou pilines (RrgA, RrgB et RrgC) et trois autres gènes codent pour les enzymes, appelées sortases, qui catalysent l'association covalente des pilines (SrtC-1, SrtC-2 et SrtC-3). Des modèles de formation du pilus ont été proposés suite à des études de délétion génétique, mais aucune donnée biochimique permettant d'expliquer précisément la formation du pilus au niveau biomoléculaire n'est encore disponible. L'étude individuelle des protéines impliquées dans la formation du pilus a permis la mise en évidence de ponts intramoléculaires Lys-Asn stabilisateurs présents dans chacune des pilines. De plus, la résolution cristallographique de RrgA et RrgB permet de mieux comprendre les propriétés adhésives de cette structure mais également son mécanisme d'assemblage. Comme le rôle de chacune des sortases reste imprécis, nous avons développé un système de co-expression permettant de tester toutes les combinaisons de pilines et de sortases. Celui-ci nous a permis d'identifier les spécificités de chacune des sortases, de générer des complexes covalents piline/piline mais également piline/sortase et ainsi d'obtenir des éléments clés dans la compréhension de la biogenèse de cette structure

Mécanismes moléculaires de la biogenèse du pilus chez Streptococcus pneumoniae

2010-10-20Streptococcus pneumoniae is the most common cause of otitis, sinusitis, pneumonia, sepsis and meningitis. Recently, pili were identified at the surface of S. pneumoniae and were proposed to play a role in the initial step of host tissues colonisation. Six genes are involved in the pilus formation. Three of them encode structural proteins named pilins (RrgA, RrgB and RrgC), the three others encode specific enzymes called sortases, which catalyse the covalent association of the pilins (SrtC-1, SrtC-2 and SrtC-3). Pilus formation models have been proposed based on genetic studies, but no biochemical data explaining precisely the molecular process of pilus formation is yet available. The individual study of each pilin led to the identification of stabilising Lys-Asn intramolecular bonds in each protein. Moreover, the cristallographic structure of RrgA and RrgB provided precious informations regarding the adhesive properties as well as the mechanism of pilus assembly. Since the role of each sortase remains unclear, our aim was to elucidate, step by step, the molecular mechanisms involved in the pilus biogenesis. Therefore, we have developed a co-expression plateform allowing the production of the pilins subunits together with sortases in E.coli. This system allowed to decipher the substrate specificity of the sortases, to generate covalent pilin/pilin complexes, as well as pilin/sortase complexes and thus provided key information towards the understanding complex macromolecular process.Streptococcus pneumoniae est un pathogène majeur chez l'homme, responsable d'otites, de pneumonies, de septicémies et de méningites. Récemment des structures de type pilus ont été identifiées à la surface de S. pneumoniae et jouent un rôle important dans les étapes initiales de colonisation des tissus hôtes. Six gènes sont impliqués dans la formation de cette structure. Trois d'entre eux codent pour les protéines structurales ou pilines (RrgA, RrgB et RrgC) et trois autres gènes codent pour les enzymes, appelées sortases, qui catalysent l'association covalente des pilines (SrtC-1, SrtC-2 et SrtC-3). Des modèles de formation du pilus ont été proposés suite à des études de délétion génétique, mais aucune donnée biochimique permettant d'expliquer précisément la formation du pilus au niveau biomoléculaire n'est encore disponible. L'étude individuelle des protéines impliquées dans la formation du pilus a permis la mise en évidence de ponts intramoléculaires Lys-Asn stabilisateurs présents dans chacune des pilines. De plus, la résolution cristallographique de RrgA et RrgB permet de mieux comprendre les propriétés adhésives de cette structure mais également son mécanisme d'assemblage. Comme le rôle de chacune des sortases reste imprécis, nous avons développé un système de co-expression permettant de tester toutes les combinaisons de pilines et de sortases. Celui-ci nous a permis d'identifier les spécificités de chacune des sortases, de générer des complexes covalents piline/piline mais également piline/sortase et ainsi d'obtenir des éléments clés dans la compréhension de la biogenèse de cette structure

The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

International audiencePosttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori. Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity

The Sole DEAD-Box RNA helicase of the gastric pathogen helicobacter pylori Is essential for colonizationω

International audiencePresent in every kingdom of life, generally in multiple copies, DEAD-box RNA helicases are specialized enzymes that unwind RNA secondary structures. They play major roles in mRNA decay, ribosome biogenesis, and adaptation to cold temperatures. Most bacteria have multiple DEAD-box helicases that present both specialized and partially redundant functions. By using phylogenomics, we revealed that the Helicobacter genus, including the major gastric pathogen H. pylori, is among the exceptions, as it encodes a sole DEAD-box RNA helicase. In H. pylori, this helicase, designated RhpA, forms a minimal RNA degradosome together with the essential RNase, RNase J, a major player in the control of RNA decay. Here, we used H. pylori as a model organism with a sole DEAD-box helicase and investigated the role of this helicase in H. pylori physiology, ribosome assembly, and during in vivo colonization. Our data showed that RhpA is dispensable for growth at 37 degrees C but crucial at 33 degrees C, suggesting an essential role of the helicase in cold adaptation. Moreover, we found that a.rhpA mutant was impaired in motility and deficient in colonization of the mouse model. RhpA is involved in the maturation of 16S rRNA at 37 degrees C and is associated with translating ribosomes. At 33 degrees C, RhpA is, in addition, recruited to individual ribosomal subunits. Finally, via its role in the RNA degradosome, RhpA directs the regulation of the expression of its partner, RNase J. RhpA is thus a multifunctional enzyme that, in H. pylori, plays a central role in gene regulation and in the control of virulence. IMPORTANCE We present the results of our study on the role of RhpA, the sole DEAD-box RNA helicase encoded by the major gastric pathogen Helicobacter pylori. We observed that all the Helicobacter species possess such a sole helicase, in contrast to most free-living bacteria. RhpA is not essential for growth of H. pylori under normal conditions. However, deletion of rhpA leads to a motility defect and to total inhibition of the ability of H. pylori to colonize a mouse model. We also demonstrated that this helicase encompasses most of the functions of its specialized orthologs described so far. We found that RhpA is a key element of the bacterial adaptation to colder temperatures and plays a minor role in ribosome biogenesis. Finally, RhpA regulates transcription of the rnj gene encoding RNase J, its essential partner in the minimal H. pylori RNA degradosome, and thus plays a crucial role in the control of RNA decay

A peptide of a type I toxin−antitoxin system induces Helicobacter pylori morphological transformation from spiral shape to coccoids

Publisher: National Academy of Sciences Section: Biological SciencesToxin−antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins’ biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin−antitoxin system (AapA1−IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori. We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5′-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae*

Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process