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1.
Antimicrob Agents Chemother ; 68(5): e0005724, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38526080

RESUMEN

Enterohemorrhagic Escherichia coli causes watery to bloody diarrhea, which may progress to hemorrhagic colitis and hemolytic-uremic syndrome. While early studies suggested that antibiotic treatment may worsen the pathology of an enterohemorrhagic Escherichia coli (EHEC) infection, recent work has shown that certain non-Shiga toxin-inducing antibiotics avert disease progression. Unfortunately, both intestinal bacterial infections and antibiotic treatment are associated with dysbiosis. This can alleviate colonization resistance, facilitate secondary infections, and potentially lead to more severe illness. To address the consequences in the context of an EHEC infection, we used the established mouse infection model organism Citrobacter rodentium ϕstx2dact and monitored changes in fecal microbiota composition during infection and antibiotic treatment. C. rodentium ϕstx2dact infection resulted in minor changes compared to antibiotic treatment. The infection caused clear alterations in the microbial community, leading mainly to a reduction of Muribaculaceae and a transient increase in Enterobacteriaceae distinct from Citrobacter. Antibiotic treatments of the infection resulted in marked and distinct variations in microbiota composition, diversity, and dispersion. Enrofloxacin and trimethoprim/sulfamethoxazole, which did not prevent Shiga toxin-mediated organ damage, had the least disruptive effects on the intestinal microbiota, while kanamycin and tetracycline, which rapidly cleared the infection without causing organ damage, caused a severe reduction in diversity. Kanamycin treatment resulted in the depletion of all but Bacteroidetes genera, whereas tetracycline effects on Clostridia were less severe. Together, these data highlight the need to address the impact of individual antibiotics in the clinical care of life-threatening infections and consider microbiota-regenerating therapies.IMPORTANCEUnderstanding the impact of antibiotic treatment on EHEC infections is crucial for appropriate clinical care. While discouraged by early studies, recent findings suggest certain antibiotics can impede disease progression. Here, we investigated the impact of individual antibiotics on the fecal microbiota in the context of an established EHEC mouse model using C. rodentium ϕstx2dact. The infection caused significant variations in the microbiota, leading to a transient increase in Enterobacteriaceae distinct from Citrobacter. However, these effects were minor compared to those observed for antibiotic treatments. Indeed, antibiotics that most efficiently cleared the infection also had the most detrimental effect on the fecal microbiota, causing a substantial reduction in microbial diversity. Conversely, antibiotics showing adverse effects or incomplete bacterial clearance had a reduced impact on microbiota composition and diversity. Taken together, our findings emphasize the delicate balance required to weigh the harmful effects of infection and antibiosis in treatment.


Asunto(s)
Antibacterianos , Citrobacter rodentium , Infecciones por Enterobacteriaceae , Heces , Microbioma Gastrointestinal , Ratones Endogámicos C57BL , Animales , Citrobacter rodentium/efectos de los fármacos , Ratones , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/efectos adversos , Heces/microbiología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Combinación Trimetoprim y Sulfametoxazol/uso terapéutico , Combinación Trimetoprim y Sulfametoxazol/farmacología , Escherichia coli Enterohemorrágica/efectos de los fármacos , Enrofloxacina/farmacología , Enrofloxacina/uso terapéutico , Femenino , Modelos Animales de Enfermedad , Disbiosis/microbiología
2.
PLoS Pathog ; 16(1): e1008184, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31951643

RESUMEN

Frequent transitions of bacterial pathogens between their warm-blooded host and external reservoirs are accompanied by abrupt temperature shifts. A temperature of 37°C serves as reliable signal for ingestion by a mammalian host, which induces a major reprogramming of bacterial gene expression and metabolism. Enteric Yersiniae are Gram-negative pathogens accountable for self-limiting gastrointestinal infections. Among the temperature-regulated virulence genes of Yersinia pseudotuberculosis is cnfY coding for the cytotoxic necrotizing factor (CNFY), a multifunctional secreted toxin that modulates the host's innate immune system and contributes to the decision between acute infection and persistence. We report that the major determinant of temperature-regulated cnfY expression is a thermo-labile RNA structure in the 5'-untranslated region (5'-UTR). Various translational gene fusions demonstrated that this region faithfully regulates translation initiation regardless of the transcription start site, promoter or reporter strain. RNA structure probing revealed a labile stem-loop structure, in which the ribosome binding site is partially occluded at 25°C but liberated at 37°C. Consistent with translational control in bacteria, toeprinting (primer extension inhibition) experiments in vitro showed increased ribosome binding at elevated temperature. Point mutations locking the 5'-UTR in its 25°C structure impaired opening of the stem loop, ribosome access and translation initiation at 37°C. To assess the in vivo relevance of temperature control, we used a mouse infection model. Y. pseudotuberculosis strains carrying stabilized RNA thermometer variants upstream of cnfY were avirulent and attenuated in their ability to disseminate into mesenteric lymph nodes and spleen. We conclude with a model, in which the RNA thermometer acts as translational roadblock in a two-layered regulatory cascade that tightly controls provision of the CNFY toxin during acute infection. Similar RNA structures upstream of various cnfY homologs suggest that RNA thermosensors dictate the production of secreted toxins in a wide range of pathogens.


Asunto(s)
Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/metabolismo , Infecciones por Yersinia pseudotuberculosis/microbiología , Yersinia pseudotuberculosis/metabolismo , Regiones no Traducidas 5' , Animales , Toxinas Bacterianas/química , Femenino , Humanos , Secuencias Invertidas Repetidas , Ratones , Ratones Endogámicos BALB C , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/genética , Temperatura , Virulencia , Yersinia pseudotuberculosis/química , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad
3.
PLoS Pathog ; 16(9): e1008552, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32966346

RESUMEN

Type VI secretion systems (T6SSs) are complex macromolecular injection machines which are widespread in Gram-negative bacteria. They are involved in host-cell interactions and pathogenesis, required to eliminate competing bacteria, or are important for the adaptation to environmental stress conditions. Here we identified regulatory elements controlling the T6SS4 of Yersinia pseudotuberculosis and found a novel type of hexameric transcription factor, RovC. RovC directly interacts with the T6SS4 promoter region and activates T6SS4 transcription alone or in cooperation with the LysR-type regulator RovM. A higher complexity of regulation was achieved by the nutrient-responsive global regulator CsrA, which controls rovC expression on the transcriptional and post-transcriptional level. In summary, our work unveils a central mechanism in which RovC, a novel key activator, orchestrates the expression of the T6SS weapons together with a global regulator to deploy the system in response to the availability of nutrients in the species' native environment.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Sistemas de Secreción Tipo VI/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Conformación Proteica , Estrés Fisiológico , Sistemas de Secreción Tipo VI/química , Sistemas de Secreción Tipo VI/genética , Yersinia pseudotuberculosis/genética
4.
PLoS Pathog ; 15(6): e1007813, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31173606

RESUMEN

Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact.


Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Endorribonucleasas/metabolismo , ARN Bacteriano/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Endorribonucleasas/genética , Humanos , ARN Bacteriano/genética , Sistemas de Secreción Tipo III/genética , Yersinia pseudotuberculosis/genética
5.
Curr Top Microbiol Immunol ; 427: 11-33, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31218505

RESUMEN

Type III secretion systems (T3SSs) are utilized by numerous Gram-negative bacteria to efficiently interact with host cells and manipulate their function. Appropriate expression of type III secretion genes is achieved through the integration of multiple control elements and regulatory pathways that ultimately coordinate the activity of a central transcriptional activator usually belonging to the AraC/XylS family. Although several regulatory elements are conserved between different species and families, each pathogen uses a unique set of control factors and mechanisms to adjust and optimize T3SS gene expression to the need and lifestyle of the pathogen. This is reflected by the complex set of sensory systems and diverse transcriptional, post-transcriptional and post-translational control strategies modulating T3SS expression in response to environmental and intrinsic cues. Whereas some pathways regulate solely the T3SS, others coordinately control expression of one or multiple T3SSs together with other virulence factors and fitness traits on a global scale. Over the past years, several common regulatory themes emerged, e.g., environmental control by two-component systems and carbon metabolism regulators or coupling of T3SS induction with host cell contact/translocon-effector secretion. One of the remaining challenges is to resolve the understudied post-transcriptional regulation of T3SS and the dynamics of the control process.


Asunto(s)
Regulación Bacteriana de la Expresión Génica , Transcripción Genética , Sistemas de Secreción Tipo III/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Factores de Transcripción/metabolismo , Factores de Virulencia
6.
PLoS Pathog ; 14(2): e1006858, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29390040

RESUMEN

Gastrointestinal infections caused by enteric yersiniae can become persistent and complicated by relapsing enteritis and severe autoimmune disorders. To establish a persistent infection, the bacteria have to cope with hostile surroundings when they transmigrate through the intestinal epithelium and colonize underlying gut-associated lymphatic tissues. How the bacteria gain a foothold in the face of host immune responses is poorly understood. Here, we show that the CNFY toxin, which enhances translocation of the antiphagocytic Yop effectors, induces inflammatory responses. This results in extensive tissue destruction, alteration of the intestinal microbiota and bacterial clearance. Suppression of CNFY function, however, increases interferon-γ-mediated responses, comprising non-inflammatory antimicrobial activities and tolerogenesis. This process is accompanied by a preterm reprogramming of the pathogen's transcriptional response towards persistence, which gives the bacteria a fitness edge against host responses and facilitates establishment of a commensal-type life style.


Asunto(s)
Toxinas Bacterianas/genética , Eliminación de Gen , Inflamación/genética , Factores de Virulencia/genética , Infecciones por Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/genética , Animales , Ciego/microbiología , Progresión de la Enfermedad , Femenino , Gastroenteritis/genética , Gastroenteritis/microbiología , Enfermedades Gastrointestinales/genética , Enfermedades Gastrointestinales/microbiología , Microbioma Gastrointestinal/fisiología , Inflamación/microbiología , Ratones , Ratones Endogámicos BALB C , Organismos Modificados Genéticamente , Yersinia pseudotuberculosis/patogenicidad , Infecciones por Yersinia pseudotuberculosis/patología
7.
Proc Natl Acad Sci U S A ; 114(5): E791-E800, 2017 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-28096329

RESUMEN

Pathogenic bacteria need to rapidly adjust their virulence and fitness program to prevent eradication by the host. So far, underlying adaptation processes that drive pathogenesis have mostly been studied in vitro, neglecting the true complexity of host-induced stimuli acting on the invading pathogen. In this study, we developed an unbiased experimental approach that allows simultaneous monitoring of genome-wide infection-linked transcriptional alterations of the host and colonizing extracellular pathogens. Using this tool for Yersinia pseudotuberculosis-infected lymphatic tissues, we revealed numerous alterations of host transcripts associated with inflammatory and acute-phase responses, coagulative activities, and transition metal ion sequestration, highlighting that the immune response is dominated by infiltrating neutrophils and elicits a mixed TH17/TH1 response. In consequence, the pathogen's response is mainly directed to prevent phagocytic attacks. Yersinia up-regulates the gene and expression dose of the antiphagocytic type III secretion system (T3SS) and induces functions counteracting neutrophil-induced ion deprivation, radical stress, and nutritional restraints. Several conserved bacterial riboregulators were identified that impacted this response. The strongest influence on virulence was found for the loss of the carbon storage regulator (Csr) system, which is shown to be essential for the up-regulation of the T3SS on host cell contact. In summary, our established approach provides a powerful tool for the discovery of infection-specific stimuli, induced host and pathogen responses, and underlying regulatory processes.


Asunto(s)
Interacciones Huésped-Patógeno/genética , Transcriptoma , Infecciones por Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/genética , Animales , Femenino , Ratones Endogámicos BALB C , Ganglios Linfáticos Agregados/metabolismo , Ganglios Linfáticos Agregados/microbiología , ARN Mensajero/genética , Análisis de Secuencia de ARN , Factores de Virulencia/genética , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/fisiología , Infecciones por Yersinia pseudotuberculosis/inmunología
8.
PLoS Pathog ; 12(12): e1006091, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28006011

RESUMEN

Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling accurately mirrored the experimental behavior and predicted that the thermoresponsiveness of this sophisticated bistable switch is mainly determined by the thermo-triggered increase of RovA proteolysis. We further observed RovA ON and OFF subpopulations of Y. pseudotuberculosis in the Peyer's patches and caecum of infected mice, and that changes in the RovA ON/OFF cell ratio reduce tissue colonization and overall virulence. This points to a bet-hedging strategy in which the thermoresponsive bistable switch plays a key role in adapting the bacteria to the fluctuating conditions encountered as they pass through the host's intestinal epithelium and suggests novel strategies for the development of antimicrobial therapies.


Asunto(s)
Proteínas Bacterianas/metabolismo , Factores de Transcripción/metabolismo , Factores de Virulencia/metabolismo , Infecciones por Yersinia pseudotuberculosis/parasitología , Yersinia pseudotuberculosis/patogenicidad , Animales , Western Blotting , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Femenino , Citometría de Flujo , Ratones , Ratones Endogámicos BALB C , Temperatura , Imagen de Lapso de Tiempo , Virulencia
9.
PLoS Genet ; 11(3): e1005087, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25816203

RESUMEN

One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y. pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated 'acetate switch', which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles.


Asunto(s)
Proteína Receptora de AMP Cíclico/genética , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica , Regulón/genética , Yersinia pseudotuberculosis/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Regulación Fúngica de la Expresión Génica , Interacción Gen-Ambiente , ARN sin Sentido/genética , ARN sin Sentido/aislamiento & purificación , Riboswitch/genética , Temperatura , Sitio de Iniciación de la Transcripción , Yersinia pseudotuberculosis/crecimiento & desarrollo , Yersinia pseudotuberculosis/patogenicidad
10.
PLoS Pathog ; 11(1): e1004600, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25590628

RESUMEN

We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.


Asunto(s)
Análisis de Secuencia de ARN/métodos , Virulencia/genética , Infecciones por Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad , Animales , Ciego/inmunología , Ciego/microbiología , Ciego/patología , Femenino , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Ratones , Análisis por Micromatrices , Microbiota/inmunología , ARN Bacteriano/genética , Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/patología
11.
RNA Biol ; 14(5): 471-487, 2017 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-27442607

RESUMEN

Enteric pathogens of the family Enterobacteriaceae colonize various niches within animals and humans in which they compete with intestinal commensals and are attacked by the host immune system. To survive these hostile environments they possess complex, multilayer regulatory networks that coordinate the control of virulence factors, host-adapted metabolic functions and stress resistance. An important part of these intricate control networks are RNA-based control systems that enable the pathogen to fine-tune its responses. Recent next-generation sequencing approaches revealed a large repertoire of conserved and species-specific riboregulators, including numerous cis- and trans-acting non-coding RNAs, sensory RNA elements (RNA thermometers, riboswitches), regulatory RNA-binding proteins and RNA degrading enzymes which regulate colonization factors, toxins, host defense processes and virulence-relevant physiological and metabolic processes. All of which are important cues for pathogens to sense and respond to fluctuating conditions during the infection. This review covers infection-relevant riboregulators of E. coli, Salmonella, Shigella and Yersinia, highlights their versatile regulatory mechanisms, complex target regulons and functions, and discusses emerging topics and future challenges to fully understand and exploit RNA-based control to combat bacterial infections.


Asunto(s)
Enterobacteriaceae/genética , Enterobacteriaceae/patogenicidad , Regulación Bacteriana de la Expresión Génica/genética , ARN Bacteriano , Animales , Infecciones por Enterobacteriaceae , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interacciones Huésped-Patógeno , Humanos , Ratones , Estabilidad del ARN , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ribonucleasas/metabolismo , Riboswitch , Virulencia/genética , Factores de Virulencia/genética
12.
J Bacteriol ; 198(20): 2876-86, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27501981

RESUMEN

UNLABELLED: The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and ΔtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ΔtatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCE: In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.


Asunto(s)
Proteínas Bacterianas/genética , Sistema de Translocación de Arginina Gemela/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Fenotipo , Transporte de Proteínas , Transcriptoma , Sistema de Translocación de Arginina Gemela/genética , Yersinia pseudotuberculosis/genética
13.
J Biol Chem ; 289(43): 30114-32, 2014 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-25164818

RESUMEN

Despite our increasing knowledge of the specific pathogenicity factors in bacteria, the contribution of metabolic processes to virulence is largely unknown. Here, we elucidate a tight connection between pathogenicity and core metabolism in the enteric pathogen Yersinia pseudotuberculosis by integrated transcriptome and [(13)C]fluxome analysis of the wild type and virulence-regulator mutants. During aerobic growth on glucose, Y. pseudotuberculosis reveals an unusual flux distribution with a high level of secreted pyruvate. The absence of the transcriptional and post-transcriptional regulators RovA, CsrA, and Crp strongly perturbs the fluxes of carbon core metabolism at the level of pyruvate metabolism and the tricarboxylic acid (TCA) cycle, and these perturbations are accompanied by transcriptional changes in the corresponding enzymes. Knock-outs of regulators of this metabolic branch point and of its central enzyme, pyruvate kinase (ΔpykF), result in mutants with significantly reduced virulence in an oral mouse infection model. In summary, our work identifies the pyruvate-TCA cycle node as a focal point for controlling the host colonization and virulence of Yersinia.


Asunto(s)
Ciclo del Ácido Cítrico , Piruvatos/metabolismo , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/patogenicidad , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomasa , Ciclo del Ácido Cítrico/efectos de los fármacos , Ciclo del Ácido Cítrico/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Glucosa/farmacología , Hierro/farmacología , Análisis de Flujos Metabólicos , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Mutación/genética , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Virulencia/efectos de los fármacos , Virulencia/genética , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/crecimiento & desarrollo , Infecciones por Yersinia pseudotuberculosis/microbiología , Infecciones por Yersinia pseudotuberculosis/patología
14.
PLoS Pathog ; 8(2): e1002518, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22359501

RESUMEN

Expression of all Yersinia pathogenicity factors encoded on the virulence plasmid, including the yop effector and the ysc type III secretion genes, is controlled by the transcriptional activator LcrF in response to temperature. Here, we show that a protein- and RNA-dependent hierarchy of thermosensors induce LcrF synthesis at body temperature. Thermally regulated transcription of lcrF is modest and mediated by the thermo-sensitive modulator YmoA, which represses transcription from a single promoter located far upstream of the yscW-lcrF operon at moderate temperatures. The transcriptional response is complemented by a second layer of temperature-control induced by a unique cis-acting RNA element located within the intergenic region of the yscW-lcrF transcript. Structure probing demonstrated that this region forms a secondary structure composed of two stemloops at 25°C. The second hairpin sequesters the lcrF ribosomal binding site by a stretch of four uracils. Opening of this structure was favored at 37°C and permitted ribosome binding at host body temperature. Our study further provides experimental evidence for the biological relevance of an RNA thermometer in an animal model. Following oral infections in mice, we found that two different Y. pseudotuberculosis patient isolates expressing a stabilized thermometer variant were strongly reduced in their ability to disseminate into the Peyer's patches, liver and spleen and have fully lost their lethality. Intriguingly, Yersinia strains with a destabilized version of the thermosensor were attenuated or exhibited a similar, but not a higher mortality. This illustrates that the RNA thermometer is the decisive control element providing just the appropriate amounts of LcrF protein for optimal infection efficiency.


Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , ARN Bacteriano/genética , Transactivadores/genética , Yersiniosis/genética , Yersinia/genética , Yersinia/patogenicidad , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Femenino , Técnicas de Inactivación de Genes , Genes Bacterianos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Temperatura , Virulencia/genética , Factores de Virulencia/genética
15.
J Biol Chem ; 287(43): 35796-803, 2012 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-22936808

RESUMEN

Pathogens often rely on thermosensing to adjust virulence gene expression. In yersiniae, important virulence-associated traits are under the control of the master regulator RovA, which uses a built-in thermosensor to control its activity. Thermal upshifts encountered upon host entry induce conformational changes in the RovA dimer that attenuate DNA binding and render the protein more susceptible to proteolysis. Here, we report the crystal structure of RovA in the free and DNA-bound forms and provide evidence that thermo-induced loss of RovA activity is promoted mainly by a thermosensing loop in the dimerization domain and residues in the adjacent C-terminal helix. These determinants allow partial unfolding of the regulator upon an upshift to 37 °C. This structural distortion is transmitted to the flexible DNA-binding domain of RovA. RovA contacts mainly the DNA backbone in a low-affinity binding mode, which allows the immediate release of RovA from its operator sites. We also show that SlyA, a close homolog of RovA from Salmonella with a very similar structure, is not a thermosensor and remains active and stable at 37 °C. Strikingly, changes in only three amino acids, reflecting evolutionary replacements in SlyA, result in a complete loss of the thermosensing properties of RovA and prevent degradation. In conclusion, only minor alterations can transform a thermotolerant regulator into a thermosensor that allows adjustment of virulence and fitness determinants to their thermal environment.


Asunto(s)
Proteínas Bacterianas/química , Pliegue de Proteína , Factores de Transcripción/química , Factores de Virulencia/química , Yersinia pseudotuberculosis/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Calor , Unión Proteica , Estructura Terciaria de Proteína , Proteolisis , Salmonella/química , Salmonella/genética , Salmonella/metabolismo , Salmonella/patogenicidad , Homología Estructural de Proteína , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidad
16.
Nucleic Acids Res ; 39(4): 1294-309, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20965974

RESUMEN

Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.


Asunto(s)
Enterobacteriaceae/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , ARN Pequeño no Traducido/genética , Transcripción Genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Factores de Integración del Huésped/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa Sigma 54/metabolismo , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Sintenía , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo
17.
RNA Biol ; 9(4): 379-91, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22336760

RESUMEN

This review emphasizes the function and regulation of the Csr regulatory system in the human enteropathogen Yersinia pseudotuberculosis and compares its features with the homologous Csr/Rsm systems of related pathogens. The Csr/Rsm systems of eubacteria form a complex regulatory network in which redundant non-translated Csr/Rsm-RNAs bind the RNA-binding protein CsrA/RsmA, thereby preventing its interaction with mRNA targets. The Csr system is controlled by the BarA/UvrY-type of two-component sensor-regulator systems. Apart from that, common or pathogen-specific regulators control the abundance of the Csr components. The coordinate control of virulence factors and infection-linked physiological traits by the Csr/Rsm systems helps the pathogens to adapt individually to rapidly changing conditions to which they are exposed during the different stages of an infection. As Csr/Rsm function is relevant for full virulence, it represents a target suitable for antimicrobial drug development.


Asunto(s)
Proteínas Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas de Unión al ARN/fisiología , Yersinia pseudotuberculosis/genética , Animales , Proteínas Bacterianas/genética , Interacciones Huésped-Patógeno , Humanos , Filogenia , Procesamiento Postranscripcional del ARN , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/fisiología , Proteínas de Unión al ARN/genética , Virulencia/genética , Yersinia pseudotuberculosis/patogenicidad , Yersinia pseudotuberculosis/fisiología , Infecciones por Yersinia pseudotuberculosis/microbiología
18.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 2): 81-90, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21245528

RESUMEN

In enteropathogenic Yersinia, the expression of several early-phase virulence factors such as invasin is tightly regulated in response to environmental cues. The responsible regulatory network is complex, involving several regulatory RNAs and proteins such as the LysR-type transcription regulator (LTTR) RovM. In this study, the crystal structure of the effector-binding domain (EBD) of RovM, the first LTTR protein described as being involved in virulence regulation, was determined at a resolution of 2.4 Å. Size-exclusion chromatography and comparison with structures of full-length LTTRs show that RovM is most likely to adopt a tetrameric arrangement with two distant DNA-binding domains (DBDs), causing the DNA to bend around it. Additionally, a cavity was detected in RovM which could bind small inducer molecules.


Asunto(s)
Proteínas Bacterianas/química , Factores de Transcripción/química , Yersinia pseudotuberculosis/química , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína
19.
PLoS Pathog ; 5(5): e1000435, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19468295

RESUMEN

Pathogens, which alternate between environmental reservoirs and a mammalian host, frequently use thermal sensing devices to adjust virulence gene expression. Here, we identify the Yersinia virulence regulator RovA as a protein thermometer. Thermal shifts encountered upon host entry lead to a reversible conformational change of the autoactivator, which reduces its DNA-binding functions and renders it more susceptible for proteolysis. Cooperative binding of RovA to its target promoters is significantly reduced at 37 degrees C, indicating that temperature control of rovA transcription is primarily based on the autoregulatory loop. Thermally induced reduction of DNA-binding is accompanied by an enhanced degradation of RovA, primarily by the Lon protease. This process is also subject to growth phase control. Studies with modified/chimeric RovA proteins indicate that amino acid residues in the vicinity of the central DNA-binding domain are important for proteolytic susceptibility. Our results establish RovA as an intrinsic temperature-sensing protein in which thermally induced conformational changes interfere with DNA-binding capacity, and secondarily render RovA susceptible to proteolytic degradation.


Asunto(s)
Proteínas Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica/fisiología , Factores de Transcripción/fisiología , Yersinia/fisiología , Proteínas Bacterianas/química , Western Blotting , Genes Bacterianos/fisiología , Reacción en Cadena de la Polimerasa , Temperatura , Factores de Transcripción/química
20.
Front Microbiol ; 12: 706934, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34413840

RESUMEN

Virulence gene expression of Yersinia pseudotuberculosis changes during the different stages of infection and this is tightly controlled by environmental cues. In this study, we show that the small protein YmoA, a member of the Hha family, is part of this process. It controls temperature- and nutrient-dependent early and later stage virulence genes in an opposing manner and co-regulates bacterial stress responses and metabolic functions. Our analysis further revealed that YmoA exerts this function by modulating the global post-transcriptional regulatory Csr system. YmoA pre-dominantly enhances the stability of the regulatory RNA CsrC. This involves a stabilizing stem-loop structure within the 5'-region of CsrC. YmoA-mediated CsrC stabilization depends on H-NS, but not on the RNA chaperone Hfq. YmoA-promoted reprogramming of the Csr system has severe consequences for the cell: we found that a mutant deficient of ymoA is strongly reduced in its ability to enter host cells and to disseminate to the Peyer's patches, mesenteric lymph nodes, liver and spleen in mice. We propose a model in which YmoA controls transition from the initial colonization phase in the intestine toward the host defense phase important for the long-term establishment of the infection in underlying tissues.

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