RESUMEN
BACKGROUND: We previously reported the identification of the aapA1/IsoA1 locus as part of a new family of toxin-antitoxin (TA) systems in the human pathogen Helicobacter pylori. AapA1 belongs to type I TA bacterial toxins, and both its mechanism of action towards the membrane and toxicity features are still unclear. METHODS: The biochemical characterization of the AapA1 toxic peptide was carried out using plasmid-borne expression and mutational approaches to follow its toxicity and localization. Biophysical properties of the AapA1 interaction with lipid membranes were studied by solution and solid-state NMR spectroscopy, plasmon waveguide resonance (PWR) and molecular modeling. RESULTS: We show that despite a low hydrophobic index, this toxin has a nanomolar affinity to the prokaryotic membrane. NMR spectroscopy reveals that the AapA1 toxin is structurally organized into three distinct domains: a positively charged disordered N-terminal domain (D), a single α-helix (H), and a basic C-terminal domain (R). The R domain interacts and destabilizes the membrane, while the H domain adopts a transmembrane conformation. These results were confirmed by alanine scanning of the minimal sequence required for toxicity. CONCLUSION: Our results have shown that specific amino acid residues along the H domain, as well as the R domain, are essential for the toxicity of the AapA1 toxin. GENERAL SIGNIFICANCE: Untangling and understanding the mechanism of action of small membrane-targeting toxins are difficult, but nevertheless contributes to a promising search and development of new antimicrobial drugs.
Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Membrana Celular/química , Infecciones por Helicobacter/genética , Helicobacter pylori/química , Alanina/química , Alanina/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Toxinas Bacterianas/genética , Membrana Celular/genética , Membrana Celular/ultraestructura , Infecciones por Helicobacter/microbiología , Helicobacter pylori/patogenicidad , Humanos , Espectroscopía de Resonancia Magnética , Conformación Proteica en Hélice alfa/genética , Dominios Proteicos/genética , Resonancia por Plasmón de Superficie , Sistemas Toxina-Antitoxina/genéticaRESUMEN
Post-transcriptional regulation plays important roles to fine-tune gene expression in bacteria. In particular, regulation of type I toxin-antitoxin (TA) systems is achieved through sophisticated mechanisms involving toxin mRNA folding. Here, we set up a genetic approach to decipher the molecular underpinnings behind the regulation of a type I TA in Helicobacter pylori. We used the lethality induced by chromosomal inactivation of the antitoxin to select mutations that suppress toxicity. We found that single point mutations are sufficient to allow cell survival. Mutations located either in the 5' untranslated region or within the open reading frame of the toxin hamper its translation by stabilizing stem-loop structures that sequester the Shine-Dalgarno sequence. We propose that these short hairpins correspond to metastable structures that are transiently formed during transcription to avoid premature toxin expression. This work uncovers the co-transcriptional inhibition of translation as an additional layer of TA regulation in bacteria.
Asunto(s)
Toxinas Bacterianas/genética , Helicobacter pylori/metabolismo , Conformación de Ácido Nucleico , Pliegue del ARN , ARN Mensajero/química , Sistemas Toxina-Antitoxina , Toxinas Bacterianas/biosíntesis , Helicobacter pylori/genética , Helicobacter pylori/crecimiento & desarrollo , Viabilidad Microbiana , Mutación Puntual , Biosíntesis de Proteínas , ARN Mensajero/genética , Selección GenéticaRESUMEN
In most bacteria, ribosomal RNA is transcribed as a single polycistronic precursor that is first processed by RNase III. This double-stranded specific RNase cleaves two large stems flanking the 23S and 16S rRNA mature sequences, liberating three 16S, 23S and 5S rRNA precursors, which are further processed by other ribonucleases. Here, we investigate the rRNA maturation pathway of the human gastric pathogen Helicobacter pylori. This bacterium has an unusual arrangement of its rRNA genes, the 16S rRNA gene being separated from a 23S-5S rRNA cluster. We show that RNase III also initiates processing in this organism, by cleaving two typical stem structures encompassing 16S and 23S rRNAs and an atypical stem-loop located upstream of the 5S rRNA. Deletion of RNase III leads to the accumulation of a large 23S-5S precursor that is found in polysomes, suggesting that it can function in translation. Finally, we characterize a cis-encoded antisense RNA overlapping the leader of the 23S-5S rRNA precursor. We present evidence that this antisense RNA interacts with this precursor, forming an intermolecular complex that is cleaved by RNase III. This pairing induces additional specific cleavages of the rRNA precursor coupled with a rapid degradation of the antisense RNA.
Asunto(s)
Helicobacter pylori/genética , Precursores del ARN/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 5S/metabolismo , Ribonucleasa III/metabolismo , Escherichia coli/metabolismo , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos/genética , Polirribosomas/metabolismo , ARN sin Sentido , ARN Bacteriano/metabolismo , ARN Ribosómico/metabolismo , Gastropatías/microbiologíaRESUMEN
As the number of bacterial genomes and transcriptomes increases, so does the number of newly identified toxin-antitoxin (TA) systems. However, their functional characterization remains challenging, often requiring the use of overexpression vectors that can lead to misinterpretations of in vivo results. To fill this gap, we developed a systematic approach called FASTBAC-Seq (Functional AnalysiS of Toxin-Antitoxin Systems in BACteria by Deep Sequencing). Combining life/death phenotypic selection with next-generation sequencing, FASTBAC-Seq allows the rapid identification of loss-of-function (toxicity) mutations in toxin-encoding genes belonging to TA loci with nucleotide resolution. Here, we present the setup used on the first-time application of FASBACT-Seq to characterize a member of the aapA/IsoA family of type I TA systems hosted on the chromosome of the major human gastric pathogen Helicobacter pylori. We propose FASBACT-Seq as a powerful tool for the functional characterization of TA systems that can in addition uncover key elements for the understanding of gene expression regulation in bacteria.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Sistemas Toxina-Antitoxina/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Sistemas Toxina-Antitoxina/genéticaRESUMEN
Type I toxin-antitoxin (TA) systems have been identified in a wide range of bacterial genomes. Here, we report the characterization of a new type I TA system present on the chromosome of the major human gastric pathogen, Helicobacter pylori. We show that the aapA1 gene encodes a 30 amino acid peptide whose artificial expression in H. pylori induces cell death. The synthesis of this toxin is prevented by the transcription of an antitoxin RNA, named IsoA1, expressed on the opposite strand of the toxin gene. We further reveal additional layers of post-transcriptional regulation that control toxin expression: (i) transcription of the aapA1 gene generates a full-length transcript whose folding impedes translation (ii) a 3Î end processing of this message generates a shorter transcript that, after a structural rearrangement, becomes translatable (iii) but this rearrangement also leads to the formation of two stem-loop structures allowing formation of an extended duplex with IsoA1 via kissing-loop interactions. This interaction ensures both the translation inhibition of the AapA1 active message and its rapid degradation by RNase III, thus preventing toxin synthesis under normal growth conditions. Finally, a search for homologous mRNA structures identifies similar TA systems in a large number of Helicobacter and Campylobacter genomes.
Asunto(s)
Antitoxinas/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Helicobacter pylori/genética , Pliegue del ARN/genética , ARN Bacteriano/genética , Antitoxinas/biosíntesis , Antitoxinas/química , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/química , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Helicobacter pylori/química , Helicobacter pylori/patogenicidad , Humanos , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/química , Ribonucleasa III/química , Ribonucleasa III/genéticaRESUMEN
Chronic Helicobacter pylori infection provokes an inflammation of the gastric mucosa, at high risk for ulcer and cancer development. The most virulent strains harbor the cag pathogenicity island (cagPAI) encoding a type 4 secretion system, which allows delivery of bacterial effectors into gastric epithelial cells, inducing pro-inflammatory responses and phenotypic alterations reminiscent of an epithelial-to-mesenchymal transition (EMT). This study characterizes EMT features in H. pylori-infected gastric epithelial cells, and investigates their relationship with NF-κB activation. Cultured human gastric epithelial cell lines were challenged with a cagPAI+ H. pylori strain or cag isogenic mutants. Morphological changes, epithelial and mesenchymal gene expression and EMT-related microRNAs were studied. H. pylori up-regulates mesenchymal markers, including ZEB1. This transcription factor is prominently involved in the mesenchymal transition of infected cells and its up-regulation depends on cagPAI and NF-κB activation. ZEB1 expression and NF-κB activation were confirmed by immunohistochemistry in gastric mucosa from cagPAI+ H. pylori-infected patients. Gastric epithelial cell lines express high miR-200 levels, which are linked to ZEB1 in a reciprocal negative feedback loop and maintain their epithelial phenotype in non-infected conditions. However, miR-200b/c were increased upon infection, despite ZEB1 up-regulation and mesenchymal morphology. In the miR-200b-200a-429 cluster promoter, we identified a functional NF-κB binding site, recruiting NF-κB upon infection and trans-activating the microRNA cluster transcription. In conclusion, in gastric epithelial cells, cagPAI+ H. pylori activates NF-κB, which transactivates ZEB1, subsequently promoting mesenchymal transition. The unexpected N-FκB-dependent increase of miR-200 levels likely thwarts the irreversible loss of epithelial identity in that critical situation.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/genética , Helicobacter pylori/fisiología , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Biomarcadores/metabolismo , Línea Celular , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Gastritis/genética , Gastritis/metabolismo , Gastritis/microbiología , Gastritis/patología , Expresión Génica , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , MicroARNs/genética , FN-kappa B/metabolismo , Fenotipo , Factores de Transcripción/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
Besides the fact that miR-96 and miR-182 belong to the miR-182/183 cluster, their seed region (UUGGCA, nucleotides 2-7) is identical suggesting potential common properties in mRNA target recognition and cellular functions. Here, we used the mRNA encoding Glypican-3, a heparan-sulfate proteoglycan, as a model target as its short 3' untranslated region is predicted to contain one miR-96/182 site, and assessed whether it is post-transcriptionally regulated by these two microRNAs. We found that miR-96 downregulated GPC3 expression by targeting its mRNA 3'-untranslated region and interacting with the predicted site. This downregulatory effect was due to an increased mRNA degradation and depended on Argonaute-2. Despite its seed similarity with miR-96, miR-182 was unable to regulate GPC3. This differential regulation was confirmed on two other targets, FOXO1 and FN1. By site-directed mutagenesis, we demonstrated that the miRNA nucleotide 8, immediately downstream the UUGGCA seed, plays a critical role in target recognition by miR-96 and miR-182. Our data suggest that because of a base difference at miRNA position 8, these two microRNAs control a completely different set of genes and therefore are functionally independent.
Asunto(s)
Regulación de la Expresión Génica , Glipicanos/genética , MicroARNs/química , Regiones no Traducidas 3' , Emparejamiento Base , Secuencia de Bases , Línea Celular Tumoral , Células Cultivadas , Glipicanos/metabolismo , Humanos , MicroARNs/fisiología , Datos de Secuencia MolecularRESUMEN
BACKGROUND: MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest. RESULTS: Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma. CONCLUSIONS: These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections.
RESUMEN
Genome sequencing of Helicobacter pylori has revealed the potential proteins and genetic diversity of this prevalent human pathogen, yet little is known about its transcriptional organization and noncoding RNA output. Massively parallel cDNA sequencing (RNA-seq) has been revolutionizing global transcriptomic analysis. Here, using a novel differential approach (dRNA-seq) selective for the 5' end of primary transcripts, we present a genome-wide map of H. pylori transcriptional start sites and operons. We discovered hundreds of transcriptional start sites within operons, and opposite to annotated genes, indicating that complexity of gene expression from the small H. pylori genome is increased by uncoupling of polycistrons and by genome-wide antisense transcription. We also discovered an unexpected number of approximately 60 small RNAs including the epsilon-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and trans-encoded target messenger RNAs. Our approach establishes a paradigm for mapping and annotating the primary transcriptomes of many living species.
Asunto(s)
Perfilación de la Expresión Génica , Genoma Bacteriano/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , ARN Bacteriano/genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Operón/genética , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN no Traducido , Alineación de Secuencia , Transcripción Genética/genéticaRESUMEN
We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.
Asunto(s)
Regulación Viral de la Expresión Génica , Hepacivirus/genética , Biosíntesis de Proteínas , Proteínas no Estructurales Virales/genética , Regiones no Traducidas 5'/química , Línea Celular , Clonación Molecular , Biblioteca de Genes , Humanos , ARN Viral/química , Secuencias Reguladoras de Ácido RibonucleicoRESUMEN
Oligonucleotides exhibiting a strong affinity and a high specificity for RNA hairpins were obtained by in vitro selection. Such oligomers give rise to loop-loop complexes with the target hairpins: the trans-activation responsive (TAR) element of the Human Immunodeficiency virus-1 (HIV-1) or subdomains of the Hepatitis C virus (HCV) mRNA. Chemically modified derivatives of an antiTAR aptamer were shown to compete out the binding of the viral protein Tat and to selectively inhibit the in vitro TAR-dependent transcription of a reporter gene. In addition, antisense oligomers derived from sequences selected against the domain IIId of the HCV internal ribosome entry site were shown to specifically block translation both in a cell-free assay and in cultured cells.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , Biosíntesis de Proteínas/genética , ARN Viral/genética , Proteínas Virales/biosíntesis , Animales , Duplicado del Terminal Largo de VIH/genética , Hepacivirus/genética , Hepacivirus/metabolismo , Humanos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Estructura Terciaria de Proteína/genética , Proteínas Virales/genéticaRESUMEN
The hepatitis C virus (HCV) 5' untranslated region (UTR) has been extensively studied with regard to its internal ribosomal entry site (IRES) activity. In this work we present results suggesting the existence of a strong promoter activity carried by the DNA sequence corresponding to the HCV 5' UTR. This activity was not detected when the HCV 5' UTR sequence was replaced by HCV 3' UTR or poliovirus 5' UTR sequences. These results were further confirmed by using bicistronic constructions. We demonstrated the presence of an mRNA initiated in this 5' UTR sequence and located the initiation site by the 5' RACE method at nucleotide 67. Furthermore, northern experiments and flow cytometry analysis showed the unambiguous activity of such a promoter sequence in stably transfected cells. Our results strongly suggest that the data obtained using bicistronic DNA constructs carrying the HCV 5' UTR should be analyzed not only at the translational but also at the transcriptional level.
Asunto(s)
Regiones no Traducidas 5'/genética , Hepacivirus/genética , Regiones Promotoras Genéticas/genética , Regiones no Traducidas 5'/química , Secuencia de Bases , ADN Complementario/genética , Expresión Génica , Genoma Viral , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , Sitio de Iniciación de la Transcripción , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Initiation of protein synthesis on the hepatitis C virus (HCV) mRNA involves a structured element corresponding to the 5' untranslated region and constituting an internal ribosome entry site (IRES). The domain IIId of the HCV IRES, an imperfect RNA hairpin extending from nucleotides 253 to 279 of the viral mRNA, has been shown to be essential for translation and for the binding of the 40S ribosomal subunit. We investigated the properties of a series of antisense 2'-O-methyloligoribonucleotides targeted to various portions of the domain IIId. Several oligomers, 14-17 nt in length, selectively inhibited in vitro translation of a bicistronic RNA construct in rabbit reticulocyte lysate with IC(50)s <10 nM. The effect was restricted to the second cistron (the Renilla luciferase) located downstream of the HCV IRES; no effect was observed on the expression of the first cistron (the firefly luciferase) which was translated in a cap-dependent manner. Moreover, antisense 2'-O-methyloligoribonucleotides specifically competed with the 40S ribosomal subunit for binding to the IRES RNA in a filter- retention assay. The antisense efficiency of the oligonucleotides was nicely correlated to their affinity for the IIId subdomain and to their ability to displace 40S ribosomal subunit, making this process a likely explanation for in vitro inhibition of HCV-IRES-dependent translation.