RESUMEN
The environmental control of invasin (inv) expression in Yersinia enterocolitica is mediated by a regulatory network composed of negative and positive regulators of inv gene transcription. Previously, we demonstrated that OmpR, a response regulator of the two-component signal transduction pathway EnvZ/OmpR, negatively regulates inv gene expression in Y. enterocolitica O9 by direct interaction with the inv promoter region. This study was undertaken to clarify the role of OmpR in the inv regulatory circuit in which RovA protein has been shown to positively regulate inv transcription. Using ompR, rovA, and ompR rovA Y. enterocolitica mutant backgrounds we showed that the inhibitory effect of OmpR on inv transcription may be observed only when RovA is present/active in Y. enterocolitica cells. To extend our research on inv regulation we examined the effect of OmpR on rovA gene expression. Analysis of rovA-lacZ transcriptional fusion in Y. enterocolitica wild-type and ompR background indicated that OmpR does not influence rovA expression. Thus, our results indicate that OmpR influences inv expression directly via binding to the inv promoter, but not through modulation of rovA expression.
Asunto(s)
Adhesinas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Yersinia enterocolitica/genética , Adhesinas Bacterianas/genética , Fusión Artificial Génica , Proteínas Bacterianas/genética , Eliminación de Gen , Genes Reporteros , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
The OmpR regulator positively influences flagella synthesis and negatively regulates invasin expression in Yersinia enterocolitica. To determine the physiological consequences of this inverse regulation, we analyzed the effect of the ompR mutation on the ability of Y. enterocolitica Ye9 (serotype O9, biotype 2) to adhere to and invade human epithelial HEp-2 cells and to form biofilms. Cell culture assays with ompR, flhDC and inv mutant strains, which vary in their motility and invasin expression, confirmed the important contribution of flagella to the adherent-invasive abilities of Y. enterocolitica Ye9. However, the loss of motility in the ompR strain was apparently not responsible for its low adhesion ability. When the nonmotile phenotype of the ompR mutant was artificially eliminated, an elevated level of invasion, exceeding that of the wild-type strain, was observed. Confocal laser microscopy demonstrated a decrease in the biofilm formation ability of the ompR strain that was only partially correlated with its loss of motility. These data provide evidence that OmpR promotes biofilm formation in this particular strain of Y. enterocolitica, although additional OmpR-dependent factors are also required. In addition, our findings suggest that OmpR-dependent regulation of biofilm formation could be an additional aspect of OmpR regulatory function.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas , Mutación/genética , Transactivadores/metabolismo , Yersinia enterocolitica/fisiología , Adhesión Bacteriana/genética , Línea Celular , Genes Bacterianos/genética , Humanos , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismoRESUMEN
Clinical diagnosis of enteroviral infections of the central nervous system are performed by virus isolation in sensitive cell lines and RT-PCR assay. The aim of the study was evaluation these techniques for fast diagnosis meningitis caused by enteroviruses. 69 samples (cerebrospinal fluid, CSF) were collected and analised by RT-PCR reaction. 39 samples were positive (56.5%). 20 positive sample were selected and simultaneously 20 stool samples from the same patients were collected for virus isolation in sensitive cell line. Positive isolation was observed only in one CSF (5.3%) and in 9 stool samples (45%).
Asunto(s)
Enterovirus Humano C/aislamiento & purificación , Enterovirus Humano C/patogenicidad , Infecciones por Enterovirus/virología , Meningitis Viral/virología , Adolescente , Adulto , Anciano , Líquido Cefalorraquídeo/virología , ADN Viral/análisis , Enterovirus Humano C/clasificación , Infecciones por Enterovirus/líquido cefalorraquídeo , Infecciones por Enterovirus/diagnóstico , Infecciones por Enterovirus/epidemiología , Femenino , Humanos , Masculino , Meningitis Viral/líquido cefalorraquídeo , Meningitis Viral/diagnóstico , Meningitis Viral/epidemiología , Persona de Mediana Edad , Polonia , Valor Predictivo de las Pruebas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cultivo de VirusRESUMEN
Enteroviruses are important etiologic agents of many human diseases such as diarrhea, self-limiting gastroenteritis, respiratory infections, conjunctivitis, hepatitis, aseptic meningitis, encephalitis, and paralysis. The aim of this study was the evaluation of the frequency of enteroviral infections in Poland in 2008-2009. Out of 178 clinical materials tested for the presence of enteroviruses, 24 samples (13,5%) were positive. In the case of 153 samples from patients suffering from acute flaccid paralysis (AFP), positive results were obtained for 6 samples (4%). Moreover, 25 samples coming from patients with clinical symptoms caused by nonpoliomyelitic enteroviruses were analyzed, giving 18 positive results (72%). The most frequently isolated enterovirus serotypes were Coxsackie B (25%), ECHO30 (25%) i ECHO6 (21%).
Asunto(s)
ADN Viral/análisis , Enterovirus Humano C/aislamiento & purificación , Enterovirus Humano C/patogenicidad , Infecciones por Enterovirus/diagnóstico , Infecciones por Enterovirus/epidemiología , Enterovirus Humano C/clasificación , Femenino , Humanos , Masculino , Polonia/epidemiología , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Muestreo , Serotipificación , Replicación ViralRESUMEN
To show the role of MalT protein in the regulation of mal genes, encoding proteins involved in transport and metabolism of maltose/ maltodextrins in Yersinia enterocolitica, we constructed a malT mutant which was characterized by a strong reduction in maltose transport and a loss of MBP protein. We also studied the influence of MalT activity on the production of Yop proteins in Y. enterocolitica and found that the level of these virulence factors is not changed in the malT mutant. Subsequently, transcriptional fusion malT::lacZYA was applied to study the activity of malT promoter. Monitoring of beta-galactosidase activity suggests the influence of catabolic repression on malT transcription, sincethe activity of malT promoter was decreased twofold in the presence of glucose. Furthermore, Mlc protein was identified in Y. enterocolitica as a factor regulating the transcription of malT. We observed a two-fold increase in the level of malT transcription in the mlc mutant background. Moreover, overproduction of Mlc protein strongly inhibited the activity of malT promoter. Thus, the data presented in this study suggest that the level of mal gene expression in Y. enterocolitica may be regulated by two proteins: MalT, the activator of mal transcription and Mlc, the repressor of malT expression.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Maltosa/metabolismo , Proteínas Represoras/metabolismo , Yersinia enterocolitica/genética , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Clonación Molecular , Conjugación Genética , Genes Bacterianos , Prueba de Complementación Genética , Glucosa/metabolismo , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas de Unión Periplasmáticas/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética , Factores de Virulencia/metabolismo , Yersinia enterocolitica/aislamiento & purificación , Yersinia enterocolitica/patogenicidadRESUMEN
Invasin, the major adhesion and invasion factor of Yersinia enterocolitica, is encoded by the inv gene, which is regulated by growth phase and in response to a variety of environmental conditions such as temperature, pH and osmolarity. So far, three proteins, RovA, H-NS and YmoA, have been identified as factors regulating the expression of the inv gene in enteropathogenic Yersinia. Here, data from inv' : : lacZYA chromosomal gene fusion studies are presented indicating that OmpR, the response regulator of the EnvZ/OmpR two-component system, acts to negatively regulate inv expression at the transcriptional level at 25 degrees C, and that high osmolarity enhances the inhibitory effect of this protein. In a strain lacking OmpR the expression of inv at 25 degrees C was increased sixfold, but at 37 degrees C, a temperature known to repress inv expression, this effect was not observed, suggesting that temperature regulation of inv is OmpR-independent. Furthermore, the expression of inv in the ompR background was no longer responsive to increased osmolarity. Complementation with the active ompR allele restored wild-type inv expression in the ompR mutant. In silico analysis of the Y. enterocolitica O : 9 inv promoter sequence revealed the presence of an OmpR consensus binding site located in the -15 to -33 region. OmpR was able to specifically bind to a fragment of the inv promoter containing this putative binding site in electrophoretic mobility shift assays. Thus, OmpR seems to be a repressor of inv in Y. enterocolitica.