RESUMEN
Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.
Asunto(s)
Bacteriófagos/genética , Escherichia coli Enterohemorrágica/fisiología , Escherichia coli Enterohemorrágica/virología , Infecciones por Escherichia coli/microbiología , Ribonucleasas/metabolismo , Toxina Shiga/biosíntesis , Toxina Shiga/genética , Escherichia coli Enterohemorrágica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Mitomicina/farmacología , Plásmidos/genética , Ribonucleasas/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Ensayo de Placa Viral , Factores de Virulencia/genéticaRESUMEN
PURPOSE: In enterohaemorrhagic Escherichia coli (EHEC), stx1 or stx2 genes encode Shiga toxin (Stx1 or Stx2, respectively) and are carried by prophages. The production and release of both stx phages and toxin occur upon initiation of the phage lytic cycle. Phages can further disseminate stx genes by infecting naïve bacteria in the intestine. Here, the effect of RNase E deficiency on these two virulence traits was investigated. METHODOLOGY: Cultures of the EHEC strains TEA028-rne containing low versus normal RNase E levels or the parental strain (TEA028) were treated with mitomycin C (MMC) to induce the phage lytic cycle. Phages and Stx2 titres were quantified by the double-agar assay and the receptor ELISA technique, respectively. RESULTS: RNase E deficiency in MMC-treated cells significantly reduced the yield of infectious stx2 phages. Delayed cell lysis and the appearance of encapsidated phage DNA copies suggest a slow onset of the lytic cycle. However, these observations do not entirely explain the decrease of phage yields. stx1 phages were not detected under normal or deficient RNase E levels. After an initial delay, high levels of toxin were finally produced in MMC-treated cultures. CONCLUSION: RNase E scarcity reduces stx2 phage production but not toxin. Normal concentrations of RNase E are likely required for correct phage morphogenesis. Our future work will address the mechanism of RNase E action on phage morphogenesis.
Asunto(s)
Colifagos/crecimiento & desarrollo , Endorribonucleasas/metabolismo , Escherichia coli Enterohemorrágica/enzimología , Escherichia coli Enterohemorrágica/virología , Profagos/crecimiento & desarrollo , Toxina Shiga II/biosíntesis , Bacteriólisis , Colifagos/genética , Endorribonucleasas/deficiencia , Ensayo de Inmunoadsorción Enzimática , Humanos , Profagos/genética , Toxina Shiga II/análisis , Ensayo de Placa ViralRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that assembles a type III secretion system (T3SS) on its surface. The last portion of the T3SS, called the 'translocon', is composed of a filament and a pore complex that is inserted into the membrane of intestinal epithelial cells. The genes encoding the translocon (espADB) are part of the LEE4 operon. Their expression is regulated by a complex post-transcriptional mechanism that involves the processing of LEE4 mRNA by the essential endoribonuclease RNase E. Here, we report the construction of an EHEC strain (TEA028-rne) in which RNase E can be induced by adding IPTG to the culture medium. EHEC cells deficient in RNase E displayed an abnormal morphology and slower growth, in agreement with published observations in E. coli K-12. Under those conditions, EspA and EspB were produced at higher concentrations, and protein secretion still occurred. These results indicate that RNase E negatively regulates translocon protein synthesis and demonstrate the utility of E. coli strain TEA028-rne as a tool for investigating the influence of this ribonuclease on EHEC gene expression in vitro.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/biosíntesis , Endorribonucleasas/deficiencia , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/biosíntesis , Sistemas de Secreción Tipo III/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , ADN Bacteriano , Endorribonucleasas/genética , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Isopropil Tiogalactósido/farmacología , OperónRESUMEN
Enterohaemorrhagic Escherichia coli harbours a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU-rich leader region and monophosphorylated 5'-terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine-Dalgarno element (SD2) and a translatable mini-ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini-ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini-ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU-rich leader region.
Asunto(s)
Sistemas de Secreción Bacterianos/fisiología , Ectima Contagioso/metabolismo , Endorribonucleasas/metabolismo , Escherichia coli/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Elementos Ricos en Adenilato y Uridilato , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sistemas de Secreción Bacterianos/genética , Sitios de Unión , Codón/metabolismo , Ectima Contagioso/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , Ribosomas/genéticaRESUMEN
Enterohaemorrhagic Escherichia coli (EHEC) employs a type III secretion system (T3SS) to export translocator and effector proteins required for mucosal colonization. The T3SS is encoded in a pathogenicity island called the locus of enterocyte effacement (LEE) that is organized in five major operons, LEE1 to LEE5. LEE4 encodes a regulator of secretion (SepL), translocators (EspA, D and B), two chaperones (CesD2 and L0017), a T3SS component (EscF) and an effector protein (EspF). It was originally proposed that the esp transcript is transcribed from a promoter located at the end of sepL but other authors suggested that this transcript is the result of a post-transcriptional processing event. In this study, we established that the espADB mRNA is generated by post-transcriptional processing at the end of the sepL coding sequence. RNase E is the endonuclease involved in the cleavage, but the interaction of this enzyme with other proteins through its C-terminal half is dispensable. A putative transcription termination event in the cesD2 coding region would generate the 3' end of the transcript. Similar to what has been described for other processed transcripts, the cleavage of LEE4 seems a mechanism to differentially regulate SepL and Esp protein production.
Asunto(s)
Escherichia coli Enterohemorrágica/metabolismo , Operón , Procesamiento Postranscripcional del ARN , Secuencia de Aminoácidos , Endorribonucleasas/genética , Escherichia coli Enterohemorrágica/genética , Regulación Bacteriana de la Expresión Génica , Islas Genómicas , Datos de Secuencia Molecular , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.