RESUMEN

Iron is both essential for and potentially toxic to bacteria, so the precise maintenance of iron homeostasis is necessary for their survival. Our previous study indicated that in the human enteropathogen Yersinia enterocolitica, the regulator OmpR directly controls the transcription of the fur, fecA and fepA genes, encoding the ferric uptake repressor and two transporters of ferric siderophores, respectively. This study was undertaken to determine the significance of the RNA chaperone Hfq and the small RNAs OmrA and RyhB1 in the post-transcriptional control of the expression of these OmpR targets. We show that Hfq silences fur, fecA and fepA expression post-transcriptionally and negatively affects the production of FLAG-tagged Fur, FecA and FepA proteins. In addition, we found that the fur gene is under the negative control of the sRNA RyhB1, while fecA and fepA are negatively regulated by the sRNA OmrA. Finally, our data revealed that the role of OmrA results from a complex interplay of transcriptional and post-transcriptional effects in the feedback circuit between the regulator OmpR and the sRNA OmrA. Thus, the expression of fur, fecA and fepA is subject to complex transcriptional and post-transcriptional regulation in order to maintain iron homeostasis in Y. enterocolitica.

Asunto(s)

ARN Pequeño no Traducido , Yersinia enterocolitica , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Hierro/metabolismo , Homeostasis/genética , Regulación Bacteriana de la Expresión Génica

RESUMEN

In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen Yersinia enterocolitica, whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR negatively affects the level of over a dozen Ysc-Yop proteins, which constitute a type III secretion system (T3SS) that is essential for the pathogenicity of Y. enterocolitica. Here, we focused our analysis on the role of OmpR in the expression and secretion of Yops (translocators and effectors). Western blotting with anti-Yops antiserum and specific anti-YopD, -YopE and -YopH antibodies, confirmed that the production of Yops is down-regulated by OmpR with the greatest negative effect on YopD. The RT-qPCR analysis demonstrated that, while OmpR had a negligible effect on the activity of regulatory genes virF and yscM1, it highly repressed the expression of yopD. OmpR was found to bind to the promoter of the lcrGVsycD-yopBD operon, suggesting a direct regulatory effect. In addition, we demonstrated that the negative regulatory influence of OmpR on the Ysc-Yop T3SS correlated with its positive role in the expression of flhDC, the master regulator of the flagellar-associated T3SS.

Asunto(s)

Yersinia enterocolitica , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación hacia Abajo/genética , Regulación Bacteriana de la Expresión Génica , Proteómica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo

RESUMEN

In this study, we found that the loss of OmpR, the response regulator of the two-component EnvZ/OmpR system, increases the cellular level of Fur, the master regulator of iron homeostasis in Y. enterocolitica. Furthermore, we demonstrated that transcription of the fur gene from the YePfur promoter is subject to negative OmpR-dependent regulation. Four putative OmpR-binding sites (OBSs) were indicated by in silico analysis of the fur promoter region, and their removal affected OmpR-dependent fur expression. Moreover, OmpR binds specifically to the predicted OBSs which exhibit a distinct hierarchy of binding affinity. Finally, the data demonstrate that OmpR, by direct binding to the promoters of the fecA, fepA and feoA genes, involved in the iron transport and being under Fur repressor activity, modulates their expression. It seems that the negative effect of OmpR on fecA and fepA transcription is sufficient to counteract the indirect, positive effect of OmpR resulting from decreasing the Fur repressor level. The expression of feoA was positively regulated by OmpR and this mode of action seems to be direct and indirect. Together, the expression of fecA, fepA and feoA in Y. enterocolitica has been proposed to be under a complex mode of regulation involving OmpR and Fur regulators.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Proteínas Represoras/genética , Transactivadores/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Portadoras/genética , Simulación por Computador , Homeostasis , Proteínas de Unión a Hierro/genética , Regiones Promotoras Genéticas , Receptores de Superficie Celular/genética , Yersinia enterocolitica/genética

RESUMEN

Yersinia enterocolitica exhibits a dual lifestyle, existing as both a saprophyte and a pathogen colonizing different niches within a host organism. OmpR has been recognized as a regulator that controls the expression of genes involved in many different cellular processes and the virulence of pathogenic bacteria. Here, we have examined the influence of OmpR and varying temperature (26°C vs. 37°C) on the cytoplasmic proteome of Y. enterocolitica Ye9N (bio-serotype 2/O:9, low pathogenicity). Differential label-free quantitative proteomic analysis indicated that OmpR affects the cellular abundance of a number of proteins including subunits of urease, an enzyme that plays a significant role in acid tolerance and the pathogenicity of Y. enterocolitica. The impact of OmpR on the expression of urease under different growth conditions was studied in more detail by comparing urease activity and the transcription of ure genes in Y. enterocolitica strains Ye9N and Ye8N (highly pathogenic bio-serotype 1B/O:8). Urease expression was higher in strain Ye9N than in Ye8N and in cells grown at 26°C compared to 37°C. However, low pH, high osmolarity and the presence of urea did not have a clear effect on urease expression in either strain. Further analysis showed that OmpR participates in the positive regulation of three transcriptional units encoding the multi-subunit urease (ureABC, ureEF, and ureGD) in strain Ye9N, but this was not the case in strain Ye8N. Binding of OmpR to the ureABC and ureEF promoter regions was confirmed using an electrophoretic mobility shift assay, suggesting that this factor plays a direct role in regulating the transcription of these operons. In addition, we determined that OmpR modulates the expression of a ureR-like gene encoding a putative regulator of the ure gene cluster, but in the opposite manner, i.e., positively in Ye9N and negatively in Ye8N. These findings provide some novel insights into the function of OmpR in adaptation strategies of Y. enterocolitica.

RESUMEN

We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the phem-1 and weaker phem-2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the -35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the phem-1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5'-untranslated region (5'-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Receptores de Superficie Celular/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo , Hemoproteínas/metabolismo , Hierro/metabolismo , Familia de Multigenes , Operón , Proteínas Represoras/metabolismo , Yersinia enterocolitica/clasificación

RESUMEN

Oligogalacturonide (OGA)-specific porins of the KdgM family have previously been identified and characterized in enterobacterial plant pathogens. We found that deletion of the gene encoding response regulator OmpR causes the porin KdgM2 to become one of the most abundant proteins in the outer membrane of the human enteropathogen Yersinia enterocolitica. Reporter gene fusion and real-time PCR analysis confirmed that the expression of kdgM2 is repressed by OmpR. We also found that kdgM2 expression is subject to negative regulation by KdgR, a specific repressor of genes involved in the uptake and metabolism of pectin derivatives in plant pathogens. The additive effect of kdgR and ompR mutations suggested that KdgR and OmpR regulate kdgM2 expression independently. We confirmed that kdgM2 occurs in an operon with the pelP gene, encoding the periplasmic pectate lyase PelP. A pectinolytic assay showed strong upregulation of PelP production/activity in a Y. enterocolitica strain lacking OmpR and KdgR, which corroborates the repression exerted by these regulators on kdgM2. In addition, our data showed that OmpR is responsible for up regulation of the kdgM1 gene encoding the second specific oligogalacturonide porin KdgM1. This indicates the involvement of OmpR in the reciprocal regulation of both KdgM1 and KdgM2. Moreover, we demonstrated the negative impact of OmpR on kdgR transcription, which might positively affect the expression of genes of the KdgR regulon. Binding of OmpR to the promoter regions of the kdgM2-pelP-sghX operon, and kdgM1 and kdgR genes was confirmed using the electrophoretic mobility shift assay, suggesting that OmpR can directly regulate their transcription. We also found that the overexpression of porin KdgM2 increases outer membrane permeability. Thus, OmpR-mediated regulation of the KdgM porins may contribute to the fitness of Y. enterocolitica in particular local environments.

Asunto(s)

Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Porinas/metabolismo , Regulón/genética , Regulón/fisiología , Proteínas Represoras/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano , Detergentes/farmacología , Perfilación de la Expresión Génica , Genes Bacterianos/genética , Genes Reporteros/genética , Genes Reporteros/fisiología , Pruebas de Sensibilidad Microbiana , Operón/genética , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Plásmidos/genética , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Porinas/genética , Regiones Promotoras Genéticas , Especies Reactivas de Oxígeno , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genética , Eliminación de Secuencia , Transcripción Genética , Yersinia enterocolitica/genética , beta-Galactosidasa/metabolismo

RESUMEN

Enteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodelling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host.

Asunto(s)

Adhesinas Bacterianas/biosíntesis , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Receptores de Superficie Celular/biosíntesis , Transactivadores/genética , Yersinia enterocolitica/genética , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Datos de Secuencia Molecular , Concentración Osmolar , Regiones Promotoras Genéticas , Proteoma/metabolismo , Proteómica , Receptores de Superficie Celular/genética , Virulencia

RESUMEN

OmpR is a transcriptional regulator implicated in the control of various cellular processes and functions in Enterobacteriaceae. This study was undertaken to identify genes comprising the OmpR regulon in the human gastrointestinal pathogen Yersinia enterocolitica. Derivatives of an ompR-negative strain with random transposon insertions creating transcriptional fusions with the reporter gene lacZ were isolated. These were supplied with the wild-type ompR allele in trans and then screened for OmpR-dependent changes in ß-galactosidase activity. Using this strategy, five insertions in genes/operons positively regulated by OmpR and two insertions in genes negatively regulated by this protein were identified. Genetic analysis of one of these fusion strains revealed that the gene acrR, encoding transcriptional repressor AcrR is negatively regulated by OmpR. Differential analysis of membrane proteins by SDS-PAGE followed by mass spectrometry identified the protein AcrB, a component of the AcrAB-TolC multidrug efflux pump, as being positively regulated by OmpR. Analysis of the activity of the acrR and acrAB promoters using gfp fusions confirmed their OmpR-dependent repression and activation, respectively. The identification of putative OmpR-binding sites and electrophoretic mobility shift assays confirmed that this regulator binds specifically to both promoter regions with different affinity. Examination of the activity of the acrR and acrAB promoters after the exposure of cells to different chemicals showed that bile salts can act as an OmpR-independent inducer. Taken together, our findings suggest that OmpR positively controls the expression of the AcrAB-TolC efflux pump involved in the adaptive response of Y. enterocolitica O:9 to different chemical stressors, thus conferring an advantage in particular ecological niches.

Asunto(s)

Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes MDR , Proteínas de la Membrana/biosíntesis , Transactivadores/metabolismo , Yersinia enterocolitica/metabolismo , Secuencia de Bases , Sitios de Unión , Electroforesis en Gel de Poliacrilamida , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Operón Lac , Datos de Secuencia Molecular , Plásmidos/metabolismo , Regiones Promotoras Genéticas , ARN/metabolismo , Temperatura , beta-Galactosidasa/metabolismo

RESUMEN

The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in Yersinia enterocolitica O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the ompR mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of Y. enterocolitica, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (ail, ompX, ompC and flhDC) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the ompR mutant. The serum resistance phenotype of ompR seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the ompR mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of flhDC in trans. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the ompR mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of Y. enterocolitica O:9 to complement-mediated killing through remodeling of the outer membrane.

Asunto(s)

Proteínas Bacterianas/metabolismo , Yersinia enterocolitica/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Yersinia enterocolitica/genética

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/genética

Asunto(s)

Proteínas Bacterianas/fisiología , Transactivadores/fisiología , Yersinia enterocolitica/fisiología , Biopelículas , Regulación Bacteriana de la Expresión Génica , Operón , Fosforilación , Virulencia/genética , Equilibrio Hidroelectrolítico , Yersinia enterocolitica/patogenicidad

RESUMEN

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/metabolismo

RESUMEN

Flagella and invasin play important roles during the early stages of infection by the enteric pathogen Yersinia enterocolitica. Our previous study demonstrated that OmpR negatively regulates invasin gene expression at the transcriptional level. The present study focused on the role of OmpR in the regulation of flagella expression. Motility assays and microscopic observations revealed that an ompR mutant strain exhibits a non-motile phenotype due to the lack of flagella. An analysis of flhDC::lacZYA chromosomal fusions demonstrated a decrease in flhDC expression in ompR mutant cells, suggesting a role for OmpR in the positive control of flagellar master operon flhDC, which is in contrast to the negative role it plays in Escherichia coli. Moreover, high temperature or osmolarity and low pH decreased flhDC expression and OmpR was not required for the response to these factors. Evidence from an examination of the DNA binding properties of OmpR in vitro indicated that the mechanism by which OmpR regulates flhDC is direct. Electrophoretic mobility shift assays confirmed that OmpR binds specifically to the flhDC promoter region and suggested the presence of more than one OmpR-binding site. In addition, phosphorylation of OmpR by acetyl-P appeared to stimulate the binding abilities of OmpR. Together with the results of our previous studies revealing the negative role of OmpR in the regulation of invasin expression, these findings support a model in which invasion and motility might be reciprocally regulated by OmpR.

Asunto(s)

Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Locomoción , Transactivadores/metabolismo , Yersinia enterocolitica/fisiología , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Flagelos/genética , Flagelos/fisiología , Eliminación de Gen , Expresión Génica , Perfilación de la Expresión Génica , Calor , Concentración de Iones de Hidrógeno , Microscopía , Datos de Secuencia Molecular , Presión Osmótica , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/genética , Yersinia enterocolitica/genética

RESUMEN

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/patogenicidad

RESUMEN

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.

Asunto(s)

Adhesinas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Transactivadores/metabolismo , Yersinia enterocolitica/genética , Fusión Artificial Génica , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión/genética , ADN Bacteriano/genética , Ensayo de Cambio de Movilidad Electroforética , Eliminación de Gen , Genes Reporteros , Prueba de Complementación Genética , Concentración Osmolar , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Temperatura , Transactivadores/aislamiento & purificación , Transcripción Genética , beta-Galactosidasa/análisis

RESUMEN

Yersinia enterocolitica is a human pathogen that causes gastroenteric infections. Various environmental signals control the expression of the virulence factors in pathogenic Y. enterocolitica strains. OmpR, a global transcriptional regulator controls the expression of a wide spectrum of genes, some of which are required for virulence. In this study, we amplified, cloned and sequenced a Y. enterocolitica Ye9 ompR gene. Deduced amino acid sequence has been shown to have 98% homology to the Y. enterocolitica O:8, Y. pestis, S. typhi and S. enterica serovar Typhimurium OmpR proteins. Additional cell culture experiments was performed to investigate whether OmpR takes part in the virulence of Y. enterocolitica. We found that the Y. enterocolitica ompR mutant was unable to invade HeLa cells. In conclusion, we have shown that OmpR is a very highly conserved protein among enteric bacterial pathogens which plays an important role in the Y. enterocolitica virulence.

Asunto(s)

Proteínas Bacterianas/análisis , Transactivadores/análisis , Yersinia enterocolitica/patogenicidad , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Células HeLa , Humanos , Datos de Secuencia Molecular , Transactivadores/genética , Transactivadores/fisiología , Virulencia

RESUMEN

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.

Asunto(s)

Macrófagos/microbiología , Transactivadores/genética , Transactivadores/fisiología , Yersinia enterocolitica/fisiología , Yersinia enterocolitica/patogenicidad , Animales , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Línea Celular , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Concentración de Iones de Hidrógeno , Ratones , Mutagénesis , Mutación , Concentración Osmolar , Proteínas Tirosina Fosfatasas/biosíntesis , Yersinia enterocolitica/genética