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

Hierarchical regulation of the NikR-mediated nickel response in Helicobacter pylori

Nickel is an essential metal for Helicobacter pylori, as it is the co-factor of two enzymes crucial for colonization, urease and hydrogenase. Nickel is taken up by specific transporters and its intracellular homeostasis depends on nickel-binding proteins to avoid toxicity. Nickel trafficking is controlled by the Ni(II)-dependent transcriptional regulator NikR. In contrast to other NikR proteins, NikR from H. pylori is a pleiotropic regulator that depending on the target gene acts as an activator or a repressor. We systematically quantified the in vivo Ni2+-NikR response of 11 direct NikR targets that encode functions related to nickel metabolism, four activated and seven repressed genes. Among these, four targets were characterized for the first time (hpn, hpn-like, hydA and hspA) and NikR binding to their promoter regions was demonstrated by electrophoretic mobility shift assays. We found that NikR-dependent repression was generally set up at higher nickel concentrations than activation. Kinetics of the regulation revealed a gradual and temporal NikR-mediated response to nickel where activation of nickel-protection mechanisms takes place before repression of nickel uptake. Our in vivo study demonstrates, for the first time, a chronological hierarchy in the NikR-dependent transcriptional response to nickel that is coherent with the control of nickel homeostasis in H. pylori

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

Regulation de l'expression des genes centraux d'un systeme de transport d'hydrates de carbone, le systeme des phosphotransferases dependantes du PEP d'Escherichia coli K-12

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78656 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

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

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Mécanismes d'adaptation de Helicobacter pylori à l'acidité gastrique

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Study of the nickel metabolism of Helicobacter pylori

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Pathogenesis of Helicobacter pylori infection

International audienceThe original strategies developed by Helicobacter pylori to persistently colonise its host and to deregulate its cellular functions make this bacterium an outstanding model to study host-pathogen interaction and the mechanisms responsible for bacterial-induced carcinogenesis. During the last year, significant results were obtained on the role of bacterial factors essential for gastric colonisation such as spiral shape maintenance, orientation through chemotaxis and the formation of bacteria clonal population islands inside the gastric glands. Particularities of the H pylori cell surface, a structure important for immune escape, were demonstrated. New insights in the bacterial stress response revealed the importance of DNA methylation-mediated regulation. Further findings were reported on H pylori components that mediate natural transformation and mechanisms of bacterial DNA horizontal transfer which maintain a high level of H pylori genetic variability. Within-host evolution was found to be niche-specific and probably associated with physiological differences between the antral and oxyntic gastric mucosa.In addition, with the progress of CryoEM, high-resolution structures of the major virulence factors, VacA and CagT4SS, were obtained. The use of gastric organoid models fostered research revealing, preferential accumulation of bacteria at the site of injury during infection. Several studies further characterised the role of CagA in the oncogenic properties of H pylori, identifying the activation of novel CagA-dependent pathways, leading to the promotion of genetic instabilities, epithelial-to-mesenchymal transition and finally carcinogenesis. Recent studies also highlight that microRNA-mediated regulation and epigenetic modifications, through DNA methylation, are key events in the H pylori-induced tumorigenesis process

Nickel, an essential virulence determinant of Helicobacter pylori: Transport and trafficking pathways and their targeting by bismuth

International audienceMetal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Nickel is required for the pathogenicity of H. pylori. This bacterial pathogen colonizes the stomach of about half of the human population worldwide and is associated with gastric cancer that is responsible for 800,000 deaths per year. H. pylori possesses two nickel-enzymes that are essential for in vivo colonization, a [NiFe] hydrogenase and an abundant urease responsible for resistance to gastric acidity. Because of these two enzymes, survival of H. pylori relies on an important supply of nickel, implying tight control strategies to avoid its toxic accumulation or deprivation. H. pylori possesses original mechanisms for nickel uptake, distribution, storage and trafficking that will be discussed in this review. During evolution, acquisition of nickel transporters and specific nickel-binding proteins has been a decisive event to allow Helicobacter species to become able to colonize the stomach. Accordingly, many of the factors involved in these mechanisms are required for mouse colonization by H. pylori. These mechanisms are controlled at different levels including protein interaction networks, transcriptional, post-transcriptional and post-translational regulation. Bismuth is another metal used in combination with antibiotics to efficiently treat H. pylori infections. Although the precise mode of action of bismuth is unknown, many targets have been identified in H. pylori and there is growing evidence that bismuth interferes with the essential nickel pathways. Understanding the metal pathways will help improve treatments against H. pylori and other pathogens

Bacterial RNA Degradosomes: Molecular Machines under Tight Control

International audienceBacterial RNA degradosomes are multienzyme molecular machines that act as hubs for post-transcriptional regulation of gene expression. The ribonuclease activities of these complexes require tight regulation, as they are usually essential for cell survival while potentially destructive. Recent studies have unveiled a wide variety of regulatory mechanisms including autoregulation, post-translational modifications, and protein compartmentalization. Recently, the subcellular organization of bacterial RNA degradosomes was found to present similarities with eukaryotic messenger ribonucleoprotein (mRNP) granules, membraneless compartments that are also involved in mRNA and protein storage and/or mRNA degradation. In this review, we present the current knowledge on the composition and targets of RNA degradosomes, the most recent developments regarding the regulation of these machineries, and their similarities with the eukaryotic mRNP granules