RESUMEN

The majority of Escherichia coli strains are harmless symbionts in the intestinal tract. However, there are several pathogenic forms, which are responsible for various diseases in humans and live stock. In this review we discuss the interactions between Shiga toxin-producing E. coli and enteropathogenic E. coli and their target host cells, describing their strategies to activate specific cellular signalling pathways which lead to subversion of critical physiological functions. We mainly concentrate on those pathogenic mechanisms that are dependent on a functional type III secretion system, but we also briefly discuss additional factors that contribute to the specific pathogenic profiles of Shiga toxin-producing E. coli and enreropathogenic E. coli.

Asunto(s)

Infecciones por Escherichia coli/fisiopatología , Escherichia coli/patogenicidad , Intestinos/microbiología , Toxinas Shiga/biosíntesis , Transducción de Señal , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Humanos , Intestinos/citología , Virulencia

RESUMEN

The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sepL start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions -5 and -36. Translational fusions using lacZ as a reporter gene demonstrated that sepL is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37 degrees C, a nutrient-rich environment, high osmolarity, and the presence of Mn(2+). Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sepL mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sepL gene in trans. The EDL933DeltasepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sepL in trans. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.

Asunto(s)

Proteínas Bacterianas/genética , Escherichia coli O157/genética , Proteínas de Escherichia coli , Genes Bacterianos , Actinas/análisis , Actinas/metabolismo , Secuencia de Aminoácidos , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Codón , Escherichia coli O157/metabolismo , Escherichia coli O157/patogenicidad , Eliminación de Gen , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Operón , Regiones Promotoras Genéticas , Transcripción Genética

RESUMEN

The Pas protein plays a key role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC), being required for the secretion of the Esp proteins. Here, the transcriptional regulation of the pas gene was analyzed through the construction of a pas::lacZ translational fusion. When bacteria were grown in Luria Bertani medium or tissue culture medium supplemented with HEPES, a bimodal activation curve was observed. The early phase of induction was not significantly modified by the incubation temperature (either 25 or 37 degrees C), whereas the second phase, which overlaps with the late exponential growth phase, was enhanced at 37 degrees C. The early phase was also stimulated by growth on tissue culture medium and by the addition of Ca(2+), Mn(2+)or Mg(2+) to the M9-glucose minimal medium. Primer extension analysis showed the presence of two major starts of transcription, which were located 58 and 60 bp upstream of the ATG-start codon of the Pas protein, respectively. Although these sites are very close to each other, the transcripts produced during the early induction phase mainly start on the -60 position, whereas the -58 start was activated during the second induction phase.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cloruro de Calcio/farmacología , Medios de Cultivo/farmacología , ADN Bacteriano/genética , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Operón Lac/genética , Cloruro de Magnesio/farmacología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , Temperatura , Transcripción Genética , Activación Transcripcional/efectos de los fármacos , Sistemas de Secreción Tipo III , beta-Galactosidasa/efectos de los fármacos , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo

RESUMEN

The formation of EspA-containing surface appendages in pathogenic Escherichia coli strains, both enteropathogenic E. coli (EPEC) and Shiga toxin-producing E. coli strains, is essential for critical events in the infective process, e.g., localized bacterial adherence to host cells with formation of microcolonies and induction of attaching and effacing lesions. It has been reported that EPEC mutants deficient in the production of EspD, which is encoded by the esp operon, are unable to accumulate actin underneath adherent bacteria but exhibit an attachment similar to that of the wild type. Here, we report the construction and characterization of an in-frame espD deletion mutant of the enterohemorrhagic E. coli (EHEC) strain EDL933. In contrast to what was observed in EPEC mutants, the EDL933 espD mutant not only lacked the capacity to accumulate actin but also exhibited an impaired attachment to HeLa cells. The synthesis of the EspD protein was also essential for the formation of EspA-containing filaments. Finally, localization studies demonstrated that the EspD protein is transferred to the cytoplasm and integrated into the cytoplasmic membranes of infected cells. These results help to elucidate the underlying molecular events in infections caused by EHEC.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Membrana Celular/metabolismo , Membrana Celular/microbiología , ADN Bacteriano , Escherichia coli/patogenicidad , Escherichia coli/ultraestructura , Eliminación de Gen , Células HeLa , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN

RESUMEN

We have determined that the genes encoding the secreted proteins EspA, EspD, and EspB of enterohemorrhagic Escherichia coli (EHEC) are organized in a single operon. The esp operon is controlled by a promoter located 94 bp upstream from the ATG start codon of the espA gene. The promoter is activated in the early logarithmic growth phase, upon bacterial contact with eukaryotic cells and in response to Ca2+, Mn2+, and HEPES. Transcription of the esp operon seems to be switched off in tightly attached bacteria. The activation process is regulated by osmolarity (induction at high osmolarities), modulated by temperature, and influenced by the degree of DNA supercoiling. Transcription is sigmaS dependent, and the H-NS protein contributes to its fine tuning. Identification of the factors involved in activation of the esp operon and the signals responsible for modulation may facilitate understanding of the underlying molecular events leading to sequential expression of virulence factors during natural infections caused by EHEC.

Asunto(s)

Proteínas Bacterianas/genética , Escherichia coli O157/genética , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Operón/genética , Transcripción Genética/genética , Secuencia de Aminoácidos , Adhesión Bacteriana , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Secuencia de Bases , Calcio/farmacología , ADN Superhelicoidal/genética , ADN Superhelicoidal/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/crecimiento & desarrollo , Escherichia coli O157/patogenicidad , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/genética , HEPES/farmacología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Manganeso/farmacología , Datos de Secuencia Molecular , Concentración Osmolar , Regiones Promotoras Genéticas/genética , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Factor sigma/genética , Factor sigma/fisiología , Temperatura , Transcripción Genética/efectos de los fármacos

RESUMEN

Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.

Asunto(s)

Benzaldehídos/metabolismo , Genes Bacterianos/genética , Protocatecuato-3,4-Dioxigenasa/genética , Pseudomonas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Eugenol/metabolismo , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Filogenia , Mapeo Físico de Cromosoma , Plásmidos , Protocatecuato-3,4-Dioxigenasa/metabolismo , Pseudomonas/genética , Pseudomonas/crecimiento & desarrollo , Análisis de Secuencia de ADN

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) induce so-called attaching and effacing lesions that enable the tight adherence of these pathogens to the gut epithelium. All of the genes necessary for this process are present in the locus of enterocyte effacement, which encodes a type III secretion system, the secreted Esp proteins and the surface protein intimin. In this study we sequenced the espA gene of STEC, generated and characterized a corresponding deletion mutant and raised EspA-specific monoclonal antibodies to analyse the functional role of this protein during infection. EspA was detected in often filament-like structures decorating all bacteria that had attached to HeLa cells. These appendages were especially prominent on bacteria that had not yet induced the formation of actin pedestals, indicating that they mediate the initial contact of STEC to their target cells. Consistently, a deletion of the espA gene completely abolished the capacity of such STEC mutants to bind to HeLa cells and to induce actin rearrangements. Surface appendages similar to those described in this study are also formed by Pseudomonas syringae and may represent a structural element common to many bacterial pathogens that deliver proteins into their target cells via a type III secretion system.

Asunto(s)

Actinas/metabolismo , Adhesinas Bacterianas , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Proteínas Portadoras , Proteínas de Escherichia coli , Escherichia coli/patogenicidad , Transducción de Señal , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Clonación Molecular , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli O157/genética , Escherichia coli O157/patogenicidad , Flagelina/genética , Eliminación de Gen , Células HeLa , Humanos , Macrófagos/microbiología , Ratones , Microscopía Confocal , Microscopía Electrónica de Rastreo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Análisis de Secuencia de ADN

RESUMEN

Enterohemorrhagic Escherichia coli (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic E. coli. We have identified an open reading frame (ORF) which is located upstream of the espA, espB, and espD genes on the complementary strand and which exhibits high homology to the genes spiB from Salmonella, yscD from Yersinia, and pscD from Pseudomonas. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III system required for protein secretion. Disruption of this ORF, designated pas (protein associated with secretion), abolished the secretion of Esp proteins. The mutant adhered only poorly and lost its capacities to trigger attaching and effacing activity and to invade HeLa cells. These results demonstrate that Pas is a virulence-associated factor that plays an essential role in EHEC pathogenesis.

Asunto(s)

Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli , Actinas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Escherichia coli O157/metabolismo , Escherichia coli O157/fisiología , Células HeLa , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Unión Proteica , Salmonella typhimurium/genética , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Sistemas de Secreción Tipo III

RESUMEN

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium- and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37 degrees C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium- and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/biosíntesis , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Clonación Molecular , Medios de Cultivo , Escherichia coli/genética , Escherichia coli/patogenicidad , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Toxina Shiga I , Temperatura , Virulencia