Study of the functionality of the Helicobacter pylori trans-translation components SmpB and SsrA in an heterologous system

<p>Abstract</p> <p>Background</p> <p><it>Trans</it>-translation is a ubiquitous bacterial quality control-mechanism for both transcription and translation. With its two major partners, SsrA a small stable RNA and the SmpB protein, it promotes the release of ribosomes stalled on defective mRNAs and directs the corresponding truncated proteins to degradation pathways. We have recently shown that <it>trans</it>-translation is an essential function in the gastric pathogen <it>Helicobacter pylori</it>. Our results suggested that some properties of the <it>H. pylori trans</it>-translation machinery distinguishes it from the well known system in <it>E. coli</it>. Therefore, we decided to test the functionality of the SmpB and SsrA molecules of <it>H. pylori </it>in the <it>E. coli </it>heterologous system using two established phenotypic tests.</p> <p>Results</p> <p><it>H. pylori </it>SmpB protein was found to successfully restore the <it>E. coli </it>Δ<it>smpB </it>mutant growth defect and its capacity to propagate λ<it>imm</it>^P22phage. We showed that in <it>E. coli</it>, <it>H. pylori </it>SsrA (Hp-SsrA) was stably expressed and maturated and that this molecule could restore wild type growth to the <it>E. coli </it>Δ<it>ssrA </it>mutant. Hp-SsrA mutants affected in the ribosome rescue function were not able to restore normal growth to <it>E. coli </it>Δ<it>ssrA </it>supporting a major role of ribosome rescue in this phenotype. Surprisingly, Hp-SsrA did not restore the phage λ<it>imm</it>^P22propagation capacity to the <it>E. coli </it>Δ<it>ssrA </it>mutant.</p> <p>Conclusions</p> <p>These data suggest an additional role of the tag sequence that presents specific features in Hp-SsrA. Our interpretation is that a secondary role of protein tagging in phage propagation is revealed by heterologous complementation because ribosome rescue is less efficient. In conclusion, <it>tm</it>RNAs present in all eubacteria, have coevolved with the translational machinery of their host and possess specific determinants that can be revealed by heterologous complementation studies.</p

Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V1a vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg116 (ECL1), Arg125 (top of TMIII), and Asp204 (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg125 (and Lys 125) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp125) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp204 (and Glu204) establishes TMIII contacts required for maintaining the α-hairpin fold of ECL2, which if broken (Ala204 or Arg204) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues

Trans-Translation in Helicobacter pylori: Essentiality of Ribosome Rescue and Requirement of Protein Tagging for Stress Resistance and Competence

BACKGROUND: The ubiquitous bacterial trans-translation is one of the most studied quality control mechanisms. Trans-translation requires two specific factors, a small RNA SsrA (tmRNA) and a protein co-factor SmpB, to promote the release of ribosomes stalled on defective mRNAs and to add a specific tag sequence to aberrant polypeptides to direct them to degradation pathways. Helicobacter pylori is a pathogen persistently colonizing a hostile niche, the stomach of humans. PRINCIPAL FINDINGS: We investigated the role of trans-translation in this bacterium well fitted to resist stressful conditions and found that both smpB and ssrA were essential genes. Five mutant versions of ssrA were generated in H. pylori in order to investigate the function of trans-translation in this organism. Mutation of the resume codon that allows the switch of template of the ribosome required for its release was essential in vivo, however a mutant in which this codon was followed by stop codons interrupting the tag sequence was viable. Therefore one round of translation is sufficient to promote the rescue of stalled ribosomes. A mutant expressing a truncated SsrA tag was viable in H. pylori, but affected in competence and tolerance to both oxidative and antibiotic stresses. This demonstrates that control of protein degradation through trans-translation is by itself central in the management of stress conditions and of competence and supports a regulatory role of trans-translation-dependent protein tagging. In addition, the expression of smpB and ssrA was found to be induced upon acid exposure of H. pylori. CONCLUSIONS: We conclude to a central role of trans-translation in H. pylori both for ribosome rescue possibly due to more severe stalling and for protein degradation to recover from stress conditions frequently encountered in the gastric environment. Finally, the essential trans-translation machinery of H. pylori is an excellent specific target for the development of novel antibiotics

Etude de la trans-traduction et du métabolisme des ARN chez helicobacter pylori

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Control of Clostridium difficile Physiopathology in Response to Cysteine Availability.

International audienceThe pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601

Reannotation of the genome sequence of Clostridium difficile strain 630.

International audienceA regular update of genome annotations is a prerequisite step to help maintain the accuracy and relevance of the information they contain. Five years after the first publication of the complete genome sequence of Clostridium difficile strain 630, we manually reannotated each of the coding sequences (CDSs), using a high-level annotation platform. The functions of more than 500 genes annotated previously with putative functions were reannotated based on updated sequence similarities to proteins whose functions have been recently identified by experimental data from the literature. We also modified 222 CDS starts, detected 127 new CDSs and added the enzyme commission numbers, which were not supplied in the original annotation. In addition, an intensive project was undertaken to standardize the names of genes and gene products and thus harmonize as much as possible with the HAMAP project. The reannotation is stored in a relational database that will be available on the MicroScope web-based platform (https://www.genoscope.cns.fr/agc/microscope/mage/viewer.php?S_id=752&wwwpkgdb=a78e3466ad5db29aa8fe49e8812de8a7). The original submission stored in the (International Nucleotide Sequence Database Collaboration) INSDC nucleotide sequence databases was also updated

Roles of α and β Carbonic Anhydrases of \u3cem\u3eHelicobacter pylori\u3c/em\u3e in the urease-dependent response to acidity and in colonization of the Murine Gastric Mucosa

Carbon dioxide occupies a central position in the physiology of Helicobacter pylori owing to its capnophilic nature, the large amounts of carbon dioxide produced by urease-mediated urea hydrolysis, and the constant bicarbonate supply in the stomach. Carbonic anhydrases (CA) catalyze the interconversion of carbon dioxide and bicarbonate and are involved in functions such as CO2 transport or trapping and pH homeostasis. H. pylori encodes a periplasmic α-CA (α-CA-HP) and a cytoplasmic β-CA (β-CA-HP). Single CA inactivation and double CA inactivation were obtained for five genetic backgrounds, indicating that H. pylori CA are not essential for growth in vitro. Bicarbonate-carbon dioxide exchange rates were measured by nuclear magnetic resonance spectroscopy using lysates of parental strains and CA mutants. Only the mutants defective in the α-CA-HP enzyme showed strongly reduced exchange rates. In H. pylori, urease activity is essential for acid resistance in the gastric environment. Urease activity measured using crude cell extracts was not modified by the absence of CA. With intact CA mutant cells incubated in acidic conditions (pH 2.2) in the presence of urea there was a delay in the increase in the pH of the incubation medium, a phenotype most pronounced in the absence of H. pylori α-CA. This correlated with a delay in acid activation of the urease as measured by slower ammonia production in whole cells. The role of CA in vivo was examined using the mouse model of infection with two mouse-adapted H. pylori strains, SS1 and X47-2AL. Compared to colonization by the wild-type strain, colonization by X47-2AL single and double CA mutants was strongly reduced. Colonization by SS1 CA mutants was not significantly different from colonization by wild-type strain SS1. However, when mice were infected by SS1Δ (β-CA-HP) or by a SS1 double CA mutant, the inflammation scores of the mouse gastric mucosa were strongly reduced. In conclusion, CA contribute to the urease-dependent response to acidity of H. pylori and are required for high-grade inflammation and efficient colonization by some strains

Oxytocin receptor pattern of expression in primary lung cancer and in normal human lung

In order to assess if oxytocin- and vasopressin-induced mitogenic effects detected on small-cell lung carcinoma (SCLC) cell lines could be transposed on primary SCLC, the aim of the present work was to identify mediators of these mitogenic actions on primary tumours samples. This was addressed on normal human lung tissue, on SCLC and on non-SCLC (NSCLC). Herein, we observe, in normal human lung, that OTR is colocalized with vascular endothelial cells of the lung and is not expressed by lung cells of epithelial nature. We detected mRNA amplification of V1aR, V2R and of a V2R variant. We observed that 86% of SCLC biopsies analyzed expressed at least the OTR and that 71% expressed the OTR, the V1aR and the V2R altogether. Comparatively, 50% of NSCLC biopsies tested expressed at least the OTR and 32% expressed the OTR, the V1aR and the V2R altogether. The occurrence of the V1bR/V3R is of 28 and 18% for SCLC and NSCLC, respectively. Nevertheless, for the SCLC biopsies analyzed in this study, V1bR/V3R expression correlates, in all cases, with the expression of all the other neurohypophysial peptide receptors. Our results suggest that neurohypophysial peptide antagonists may offer promise as a potential new therapeutic modality for the treatment of lung cancer expressing at least one of the neurhypophysial peptide receptor subtypes. (c) 2005 Elsevier Ireland Ltd. All rights reserved