Identifying New Variables During Infection: Proximity to the Host Epithelium and Epigenetic Programs Alter the Expression of Virulence Factors in Vibrio Cholerae

In the life cycles of facultative pathogens, the ability to transition between the external environment and the host is key to survival. These transitions are accompanied by alterations in transcriptional and proteomic profiles (Merrell et al., 2002; Schild et al., 2007; LaRocque et al., 2008). Numerous studies have investigated the changes in gene expression that occur during these environmental shifts and have identified factors that are important for survival and growth in the two disparate settings. Less is known about the changes in gene expression that result as a pathogen transitions between microenvironments within the host or even the number of such transitions that occur. A single organ or tissue may in fact constitute a wide array of strikingly different environmental conditions for an invading pathogen

Cyclic diguanylate signaling in Gram-positive bacteria

The nucleotide second messenger 3′-5′ cyclic diguanylate monophosphate (c-di-GMP) is a central regulator of the transition between motile and non-motile lifestyles in bacteria, favoring sessility. Most research investigating the functions of c-di-GMP has focused on Gram-negative species, especially pathogens. Recent work in Gram-positive species has revealed that c-di-GMP plays similar roles in Gram-positives, though the precise targets and mechanisms of regulation may differ. The majority of bacterial life exists in a surface-associated state, with motility allowing bacteria to disseminate and colonize new environments. c-di-GMP signaling regulates flagellum biosynthesis and production of adherence factors and appears to be a primary mechanism by which bacteria sense and respond to surfaces. Ultimately, c-di-GMP influences the ability of a bacterium to alter its transcriptional program, physiology and behavior upon surface contact. This review discusses how bacteria are able to sense a surface via flagella and type IV pili, and the role of c-di-GMP in regulating the response to surfaces, with emphasis on studies of Gram-positive bacteria

The RNA Domain Vc1 Regulates Downstream Gene Expression in Response to Cyclic Diguanylate in Vibrio cholerae

In many bacterial species, including the aquatic bacterium and human pathogen Vibrio cholerae, the second messenger cyclic diguanylate (c-di-GMP) modulates processes such as biofilm formation, motility, and virulence factor production. By interacting with various effectors, c-di-GMP regulates gene expression or protein function. One type of c-di-GMP receptor is the class I riboswitch, representatives of which have been shown to bind c-di-GMP in vitro. Herein, we examined the in vitro and in vivo function of the putative class I riboswitch in Vibrio cholerae, Vc1, which lies upstream of the gene encoding GbpA, a colonization factor that contributes to attachment of V. cholerae to environmental and host surfaces containing N-acetylglucosamine moieties. We provide evidence that Vc1 RNA interacts directly with c-di-GMP in vitro, and that nucleotides conserved among this class of riboswitch are important for binding. Yet the mutation of these conserved residues individually in the V. cholerae chromosome inconsistently affects the expression of gbpA and production of the GbpA protein. By isolating the regulatory function of Vc1, we show that the Vc1 element positively regulates downstream gene expression in response to c-di-GMP. Together these data suggest that the Vc1 element responds to c-di-GMP in vivo. Positive regulation of gbpA expression by c-di-GMP via Vc1 may influence the ability of V. cholerae to associate with chitin in the aquatic environment and the host intestinal environment

A novel regulator controls Clostridium difficile sporulation, motility and toxin production

Clostridium difficile is an anaerobic pathogen that forms spores which promote survival in the environment and transmission to new hosts. The regulatory pathways by which C. difficile initiates spore formation are poorly understood. We identified two factors with limited similarity to the Rap sporulation proteins of other spore-forming bacteria. In this study, we show that disruption of the gene CD3668 reduces sporulation and increases toxin production and motility. This mutant was more virulent and exhibited increased toxin gene expression in the hamster model of infection. Based on these phenotypes, we have renamed this locus rstA, for regulator of sporulation and toxins. Our data demonstrate that RstA is a bifunctional protein that upregulates sporulation through an unidentified pathway and represses motility and toxin production by influencing sigD transcription. Conserved RstA orthologs are present in other pathogenic and industrial Clostridium species and may represent a key regulatory protein controlling clostridial sporulation

The Vibrio cholerae Pst2 Phosphate Transport System Is Upregulated in Biofilms and Contributes to Biofilm-Induced Hyperinfectivity

ABSTRACT Vibrio cholerae is the causative agent of the deadly diarrheal disease cholera. As part of its life cycle, V. cholerae persists in marine environments, where it forms surface-attached communities commonly described as biofilms. Evidence indicates that these biofilms constitute the infectious form of the pathogen during outbreaks. Previous work has shown that biofilm-derived V. cholerae cells, even when fully dispersed from the biofilm matrix, are vastly more infectious than planktonic (free-living) cells. Here, we sought to identify factors that contribute to biofilm-induced hyperinfectivity in V. cholerae , and we present evidence for one aspect of the molecular basis of this phenotype. We identified proteins upregulated during growth in biofilms and determined their contributions to the hyperinfectivity phenotype. We found that PstS2, the periplasmic component of the Pst2 phosphate uptake system, was enriched in biofilms. Another gene in the pst2 locus was transcriptionally upregulated in biofilms. Using the infant mouse model, we found that mutation of two pst2 components resulted in impaired colonization. Importantly, deletion of the Pst2 inner membrane complex caused a greater colonization defect after growth in a biofilm compared to shaking culture. Based on these data, we propose that V. cholerae cells in biofilms upregulate the Pst2 system and therefore gain an advantage upon entry into the host. Further characterization of factors contributing to biofilm-induced hyperinfectivity in V. cholerae will improve our understanding of the transmission of the bacteria from natural aquatic habitats to the human host

El acuerdo comercial de Ecuador con la Unión Europea y el comportamiento de las exportaciones ecuatorianas

El comercio exterior constituye una de las actividades más influyentes en el crecimiento económico de un país; sobre esta base el Ecuador ha dado paso al acuerdo comercial con la Unión Europea, un mercado con más de 500 millones de consumidores, con alto poder adquisitivo, y que puede impulsar el crecimiento de nuestras exportaciones, con importantes efectos sobre el crecimiento económico y por ende, sobre el desarrollo y bienestar de todos los ecuatorianos, si se establecen las políticas públicas acertadas para aprovechar los beneficios y oportunidades que promete su suscripción. Este estudio parte de un breve análisis de las principales teorías de comercio exterior y se establece los factores que determinan la oferta y la demanda, cuyo abordaje permite medir la influencia del acuerdo comercial sobre el comportamiento de las exportaciones e importaciones ecuatorianas. Así también, se analiza el origen de los acuerdos comerciales, sus características y aspectos positivos y negativos, que son contrastados con los efectos evidenciados sobre la economía de Colombia y Perú, países que forman parte del acuerdo multipartes con la Unión Europea. Se profundiza en el análisis del comportamiento y principales características de las exportaciones e importaciones ecuatorianas, desembocando en la evaluación del resultado de la balanza comercial. Por último, se establecen los antecedentes de las relaciones comerciales entre Ecuador y la UE, identificando los productos que, durante el primer año de vigencia del tratado, presentan un comportamiento más cambiante, y, se analiza los resultados del IHH para exportaciones e importaciones, respaldándolos con las respuestas de los expertos entrevistados. Entre los principales resultados de la investigación, se tiene que durante el primer año de vigencia del acuerdo comercial entre Ecuador y la Unión Europea, los niveles de venta evidenciados reflejan, no solo el efecto directo de la eliminación de aranceles sino, además, la experiencia de nuestro sector exportador, lo que ha permitido que nuestros productos sean más apetecidos y demandados, condición que debe ser aprovechada para consolidar nuestros productos entre los consumidores europeos

Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile

ABSTRACT The intestinal pathogen Clostridium difficile is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States. C. difficile rates of recurrence and mortality have increased in recent years due to the emergence of so-called “hypervirulent” epidemic strains. A great deal of the basic biology of C. difficile has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for C. difficile gene regulation. However, the function(s) of TFP in C. difficile remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of C. difficile . We demonstrate that TFP contribute to C. difficile biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in C. difficile . The expression and regulation of the pilA1 pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in pilA1 expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic C. difficile strains may contribute to the different infection outcomes presented by these strains. IMPORTANCE How Clostridium difficile establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of C. difficile are likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation by C. difficile . TFP are also required for surface motility, which has not previously been demonstrated for C. difficile . Furthermore, an epidemic-associated C. difficile strain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains

Two nucleotide second messengers regulate the production of the Vibrio cholerae colonization factor GbpA

Abstract Background The nucleotide second messengers cAMP and c-di-GMP allow many bacteria, including the human intestinal pathogen Vibrio cholerae, to respond to environmental stimuli with appropriate physiological adaptations. In response to limitation of specific carbohydrates, cAMP and its receptor CRP control the transcription of genes important for nutrient acquisition and utilization; c-di-GMP controls the transition between motile and sessile lifestyles often, but not exclusively, through transcriptional mechanisms. In this study, we investigated the convergence of cAMP and c-di-GMP signaling pathways in regulating the expression of gbpA. GbpA is a colonization factor that participates in the attachment of V. cholerae to N-acetylglucosamine-containing surfaces in its native aquatic environment and the host intestinal tract. Results We show that c-di-GMP inhibits gbpA activation in a fashion independent of the known transcription factors that directly sense c-di-GMP. Interestingly, inhibition of gbpA activation by c-di-GMP only occurs during growth on non-PTS dependent nutrient sources. Consistent with this result, we show that CRP binds to the gbpA promoter in a cAMP-dependent manner in vitro and drives transcription of gbpA in vivo. The interplay between cAMP and c-di-GMP does not broadly impact the CRP-cAMP regulon, but occurs more specifically at the gbpA promoter. Conclusions These findings suggest that c-di-GMP directly interferes with the interaction of CRP-cAMP and the gbpA promoter via an unidentified regulator. The use of two distinct second messenger signaling mechanisms to regulate gbpA transcription may allow V. cholerae to finely modulate GbpA production, and therefore colonization of aquatic and host surfaces, in response to discrete environmental stimuli

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production during Stationary Phase

ABSTRACT The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile , c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability

A Nutrient-Regulated Cyclic Diguanylate Phosphodiesterase Controls Clostridium difficile Biofilm and Toxin Production During Stationary Phase

The signaling molecule cyclic diguanylate (c-di-GMP) mediates physiological adaptation to extracellular stimuli in a wide range of bacteria. The complex metabolic pathways governing c-di-GMP synthesis and degradation are highly regulated, but the specific cues that impact c-di-GMP signaling are largely unknown. In the intestinal pathogen Clostridium difficile, c-di-GMP inhibits flagellar motility and toxin production and promotes pilus-dependent biofilm formation, but no specific biological functions have been ascribed to any of the individual c-di-GMP synthases or phosphodiesterases (PDEs). Here, we report the functional and biochemical characterization of a c-di-GMP PDE, PdcA, 1 of 37 confirmed or putative c-di-GMP metabolism proteins in C. difficile 630. Our studies reveal that pdcA transcription is controlled by the nutrient-regulated transcriptional regulator CodY and accordingly increases during stationary phase. In addition, PdcA PDE activity is allosterically regulated by GTP, further linking c-di-GMP levels to nutrient availability. Mutation of pdcA increased biofilm formation and reduced toxin biosynthesis without affecting swimming motility or global intracellular c-di-GMP. Analysis of the transcriptional response to pdcA mutation indicates that PdcA-dependent phenotypes manifest during stationary phase, consistent with regulation by CodY. These results demonstrate that inactivation of this single PDE gene is sufficient to impact multiple c-di-GMP-dependent phenotypes, including the production of major virulence factors, and suggest a link between c-di-GMP signaling and nutrient availability