RESUMEN
Pseudomonas aeruginosa is a pathogen capable of colonizing virtually every human tissue. The host colonization competence and versatility of this pathogen are powered by a wide array of virulence factors necessary in different steps of the infection process. This includes factors involved in bacterial motility and attachment, biofilm formation, the production and secretion of extracellular invasive enzymes and exotoxins, the production of toxic secondary metabolites, and the acquisition of iron. Expression of these virulence factors during infection is tightly regulated, which allows their production only when they are needed. This process optimizes host colonization and virulence. In this work, we review the intricate network of transcriptional regulators that control the expression of virulence factors in P. aeruginosa, including one- and two-component systems and σ factors. Because inhibition of virulence holds promise as a target for new antimicrobials, blocking the regulators that trigger the production of virulence determinants in P. aeruginosa is a promising strategy to fight this clinically relevant pathogen.
Asunto(s)
Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Virulencia/genética , Pseudomonas aeruginosa/metabolismo , Factores de Virulencia/metabolismo , Exotoxinas/metabolismo , Percepción de Quorum , Biopelículas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones por Pseudomonas/microbiologíaRESUMEN
Cell-surface signaling (CSS) is a signal transfer system of Gram-negative bacteria that produces the activation of an extracytoplasmic function σ factor (σECF) in the cytosol in response to an extracellular signal. Activation requires the regulated and sequential proteolysis of the σECF-associated anti-σ factor, and the function of the Prc and RseP proteases. In this work, we have identified another protease that modulates CSS activity, namely the periplasmic carboxyl-terminal processing protease CtpA. CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-σ factor. Importantly, using zebrafish embryos and the A549 lung epithelial cell line as hosts, we show that mutants in the rseP and ctpA proteases of the human pathogen Pseudomonas aeruginosa are considerably attenuated in virulence while the prc mutation increases virulence likely by enhancing the production of membrane vesicles.
RESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen able to infect any human tissue. One of the reasons for its high adaptability and colonization of host tissues is its capacity of maintaining iron homeostasis through a wide array of iron acquisition and removal mechanisms. Due to their ability to cause life-threatening acute and chronic infections, especially among cystic fibrosis and immunocompromised patients, and their propensity to acquire resistance to many antibiotics, the World Health Organization (WHO) has encouraged the scientific community to find new strategies to eradicate this pathogen. Several recent strategies to battle P. aeruginosa focus on targeting iron homeostasis mechanisms, turning its greatest advantage into an exploitable weak point. In this review, we discuss the different mechanisms used by P. aeruginosa to maintain iron homeostasis and the strategies being developed to fight this pathogen by blocking these mechanisms. Among others, the use of iron chelators and mimics, as well as disruption of siderophore production and uptake, have shown promising results in reducing viability and/or virulence of this pathogen. The so-called 'Trojan-horse' strategy taking advantage of the siderophore uptake systems is emerging as an efficient method to improve delivery of antibiotics into the bacterial cells. Moreover, siderophore transporters are considered promising targets for the developing of P. aeruginosa vaccines.
Asunto(s)
Infecciones por Pseudomonas , Pseudomonas aeruginosa , Humanos , Sideróforos , Hierro , Homeostasis , Antibacterianos/uso terapéutico , Infecciones por Pseudomonas/microbiologíaRESUMEN
The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in Pseudomonas putida KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ70-dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. P. putida K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS-GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and P. putida K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.
Asunto(s)
Pseudomonas putida , Sistemas de Secreción Tipo VI , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pseudomonas putida/metabolismo , Factor sigma/genética , Familia de Multigenes , Regulación Bacteriana de la Expresión GénicaRESUMEN
Pseudomonas aeruginosa causes a wide array of life-threatening acute and chronic infections in humans. This opportunistic pathogen is metabolically highly versatile and harbors multiple virulence factors that allow infection of essentially any organ of the human body. The high capacity of this bacterium to acquire iron facilitates its versatility and is considered one of the P. aeruginosa virulence hallmarks. Iron functions as a redox cofactor of enzymes required for vital biological processes and is thus essential for all living organisms. However, in aerobic environments, iron is mainly present in its ferric form, which is insoluble and poorly bioavailable. This problem increases in the human body because, as a reaction to the infection, the host induces a "nutritional immunity" response aiming to reduce the amount of iron available for invading microorganisms. P. aeruginosa contains several mechanisms for iron acquisition including (1) production of siderophores pyoverdine and pyochelin; (2) use of xenosiderophores produced by other microorganisms; (3) direct transport of ferrous ions; and (4) utilization of host iron carriers (e.g., heme). However, although essential, iron results toxic when present in excess because it facilitates the production of reactive oxygen species (ROS) that damage bacterial cells. P. aeruginosa contains ferritins and efflux systems for iron withdrawal to avoid excess of this metal. Production of iron acquisition and removal systems is highly regulated to ensure sufficient iron for metabolic needs while preventing its toxicity. This chapter covers the different mechanisms used by P. aeruginosa to maintain iron homeostasis, which is vital for this pathogen to grow and proliferate in the host. We also highlight current strategies to block P. aeruginosa infections by disrupting iron homeostasis.
Asunto(s)
Antiinfecciosos , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/fisiología , Sideróforos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Hierro/metabolismo , Homeostasis , Factores de Virulencia/metabolismo , Hemo , Antibacterianos , Ferritinas/metabolismo , Iones , Proteínas Bacterianas/metabolismoRESUMEN
For bacteria to flourish in different niches, they need to sense signals from the environment and translate these into appropriate responses. Most bacterial signal transduction systems involve proteins that trigger the required response through the modification of gene transcription. These proteins are often produced in an inactive state that prevents their interaction with the RNA polymerase and/or the DNA in the absence of the inducing signal. Among other mechanisms, regulated proteolysis is becoming increasingly recognized as a key process in the modulation of the activity of these signal response proteins. Regulated proteolysis can either produce complete degradation or specific cleavage of the target protein, thus modifying its function. Because proteolysis is a fast process, the modulation of signaling proteins activity by this process allows for an immediate response to a given signal, which facilitates adaptation to the surrounding environment and bacterial survival. Moreover, regulated proteolysis is a fundamental process for the transmission of extracellular signals to the cytosol through the bacterial membranes. By a proteolytic mechanism known as regulated intramembrane proteolysis (RIP) transmembrane proteins are cleaved within the plane of the membrane to liberate a cytosolic domain or protein able to modify gene transcription. This allows the transmission of a signal present on one side of a membrane to the other side where the response is elicited. In this work, we review the role of regulated proteolysis in the bacterial communication with the environment through the modulation of the main bacterial signal transduction systems, namely one- and two-component systems, and alternative σ factors.
RESUMEN
The extracytoplasmic function sigma factor σVreI of the human pathogen Pseudomonas aeruginosa promotes transcription of potential virulence determinants, including secretion systems and secreted proteins. Its activity is modulated by the VreR anti-σ factor that inhibits the binding of σVreI to the RNA polymerase in the absence of a (still unknown) inducing signal. The vreI-vreR genes are expressed under inorganic phosphate (Pi) starvation, a physiological condition often encountered in the host that increases P. aeruginosa pathogenicity. However, whether or not σVreI is active in vivo during infection and contributes to the Pi starvation-induced virulence of this pathogen has not been analyzed yet. Using zebrafish embryos and a human alveolar basal epithelial cell line as P. aeruginosa hosts, we demonstrate in this work that σVreI is active during infection and that lack of σVreI considerably reduces the Pi starvation-induced virulence of this pathogen. Surprisingly, lack of the σVreI inhibitor, the VreR anti-σ factor, also diminishes the virulence of P. aeruginosa. By transcriptomic analyses we show that VreR modulates gene expression not only in a σVreI-dependent but also in a σVreI-independent manner. This includes potential virulence determinants and transcriptional regulators that could be responsible for the reduced virulence of the ΔvreR mutant.
Asunto(s)
Citoplasma/metabolismo , Células Epiteliales/microbiología , Fosfatos/metabolismo , Pseudomonas aeruginosa/metabolismo , Factor sigma/metabolismo , Virulencia , Células A549 , Animales , Proteínas Bacterianas/metabolismo , Análisis por Conglomerados , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Pulmón/metabolismo , Modelos Genéticos , Mutación , Filogenia , Alveolos Pulmonares/citología , Transducción de Señal , Transcriptoma , Factores de Virulencia/metabolismo , Pez CebraRESUMEN
Pathogens have developed several strategies to obtain iron during infection, including the use of iron-containing molecules from the host. Haem accounts for the vast majority of the iron pool in vertebrates and thus represents an important source of iron for pathogens. Using a proteomic approach, we have identified in this work a previously uncharacterized system, which we name Hxu, that together with the known Has and Phu systems, is used by the human pathogen Pseudomonas aeruginosa to respond to haem. We show that the Has and Hxu systems are functional signal transduction pathways of the cell-surface signalling class and report the mechanism triggering the activation of these signalling systems. Both signalling cascades involve an outer membrane receptor (HasR and HxuA respectively) that upon sensing haem in the extracellular medium produces the activation of an σECF factor in the cytosol. HxuA has a major role in signalling and a minor role in haem acquisition in conditions in which the HasR and PhuR receptors or other sources of iron are present. Remarkably, P. aeruginosa compensates the lack of the HasR receptor by increasing the production of HxuA, which underscores the importance of haem signalling for this pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hemo/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Hierro/metabolismo , Proteómica , Pseudomonas aeruginosa/genética , Transducción de SeñalRESUMEN
Pseudomonas bacteria are widespread and are found in soil and water, as well as pathogens of both plants and animals. The ability of Pseudomonas to colonize many different environments is facilitated by the multiple signaling systems these bacteria contain that allow Pseudomonas to adapt to changing circumstances by generating specific responses. Among others, signaling through extracytoplasmic function σ (σECF ) factors is extensively present in Pseudomonas. σECF factors trigger expression of functions required under particular conditions in response to specific signals. This manuscript reviews the phylogeny and biological roles of σECF factors in Pseudomonas, and highlights the diversity of σECF -signaling pathways of this genus in terms of function and activation. We show that Pseudomonas σECF factors belong to 16 different phylogenetic groups. Most of them are included within the iron starvation group and are mainly involved in iron acquisition. The second most abundant group is formed by RpoE-like σECF factors, which regulate the responses to cell envelope stress. Other groups controlling solvent tolerance, biofilm formation and the response to oxidative stress, among other functions, are present in lower frequency. The role of σECF factors in the virulence of Pseudomonas pathogenic species is described.
Asunto(s)
Proteínas Bacterianas/metabolismo , Espacio Extracelular/metabolismo , Pseudomonas/metabolismo , Factor sigma/metabolismo , Animales , Proteínas Bacterianas/genética , Espacio Extracelular/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Filogenia , Pseudomonas/clasificación , Pseudomonas/genética , Infecciones por Pseudomonas/microbiología , Factor sigma/genética , Transducción de SeñalRESUMEN
The type VI secretion system (T6SS) is a bacterial nanomachine used to inject effectors into prokaryotic or eukaryotic cells and is thus involved in both host manipulation and interbacterial competition. The T6SS is widespread among Gram-negative bacteria, mostly within the Proteobacterium Phylum. This secretion system is commonly found in commensal and pathogenic plant-associated bacteria. Phylogenetic analysis of phytobacterial T6SS clusters shows that they are distributed in the five main clades previously described (group 1-5). The even distribution of the system among commensal and pathogenic phytobacteria suggests that the T6SS provides fitness and colonization advantages in planta and that the role of the T6SS is not restricted to virulence. This manuscript reviews the phylogeny and biological roles of the T6SS in plant-associated bacteria, highlighting a remarkable diversity both in terms of mechanism and function.
Asunto(s)
Plantas/microbiología , Proteobacteria/metabolismo , Proteobacteria/patogenicidad , Sistemas de Secreción Tipo VI/fisiología , Proteínas Bacterianas/genética , Filogenia , Enfermedades de las Plantas/microbiología , Proteobacteria/genética , Sistemas de Secreción Tipo VI/genética , VirulenciaRESUMEN
Cell-surface signaling (CSS) is a signal transfer system that allows Gram-negative bacteria to detect environmental signals and generate a cytosolic response. These systems are composed of an outer membrane receptor that senses the inducing signal, an extracytoplasmic function sigma factor (σECF) that targets the cytosolic response by modifying gene expression and a cytoplasmic membrane anti-sigma factor that keeps the σECF in an inactive state in the absence of the signal and transduces its presence from the outer membrane to the cytosol. Although CSS systems regulate bacterial processes as crucial as stress response, iron scavenging and virulence, the exact mechanisms that drive CSS are still not completely understood. Binding of the signal to the CSS receptor is known to trigger a signaling cascade that results in the regulated proteolysis of the anti-sigma factor and the activation of the σECF in the cytosol. This study was carried out to generate new insights in the proteolytic activation of CSS σECF. We performed a random mutagenesis screen of the unique IutY protein of Pseudomonas putida, a protein that combines a cytosolic σECF domain and a periplasmic anti-sigma factor domain in a single polypeptide. In response to the presence of an iron carrier, the siderophore aerobactin, in the extracellular medium, IutY is processed by two different proteases, Prc and RseP, which results in the release and activation of the σIutY domain. Our experiments show that all IutY mutant proteins that contain periplasmic residues depend on RseP for activation. In contrast, Prc is only required for mutant variants with a periplasmic domain longer than 50 amino acids, which indicates that the periplasmic region of IutY is trimmed down to ~50 amino acids creating the RseP substrate. Moreover, we have identified several conserved residues in the CSS anti-sigma factor family of which mutation leads to constitutive activation of their cognate σECF. These findings advance our knowledge on how CSS activity is regulated by the consecutive action of two proteases. Elucidation of the exact mechanism behind CSS activation will enable the development of strategies to block CSS in pathogenic bacteria.
RESUMEN
Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines have an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyze the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, 10 T6SS effector-immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device, which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens, which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris. Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol, which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections.
Asunto(s)
Nicotiana/microbiología , Enfermedades de las Plantas/prevención & control , Pseudomonas putida/fisiología , Xanthomonas campestris/fisiología , Proteínas Bacterianas/genética , Agentes de Control Biológico , Regulación Bacteriana de la Expresión Génica , Enfermedades de las Plantas/microbiología , Pseudomonas putida/genética , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/fisiologíaRESUMEN
Gene regulation in bacteria is primarily controlled at the level of transcription initiation by modifying the affinity of the RNA polymerase (RNAP) for the promoter. This control often occurs through the substitution of the RNAP sigma (σ) subunit. Next to the primary σ factor, most bacteria contain a variable number of alternative σ factors of which the extracytoplasmic function group (σ(ECF)) is predominant. Pseudomonas aeruginosa contains nineteen σ(ECF), including the virulence regulator σ(VreI). σ(VreI) is encoded by the vreAIR operon, which also encodes a receptor-like protein (VreA) and an anti-σ factor (VreR). These three proteins form a signal transduction pathway known as PUMA3, which controls expression of P. aeruginosa virulence functions. Expression of the vreAIR operon occurs under inorganic phosphate (Pi) limitation and requires the PhoB transcription factor. Intriguingly, the genes of the σ(VreI) regulon are also expressed in low Pi despite the fact that the σ(VreI) repressor, the anti-σ factor VreR, is also produced in this condition. Here we show that although σ(VreI) is partially active under Pi starvation, maximal transcription of the σ(VreI) regulon genes requires the removal of VreR. This strongly suggests that an extra signal, probably host-derived, is required in vivo for full σ(VreI) activation. Furthermore, we demonstrate that the activity of σ(VreI) is modulated not only by VreR but also by the transcription factor PhoB. Presence of this regulator is an absolute requirement for σ(VreI) to complex the DNA and initiate transcription of the PUMA3 regulon. The potential DNA binding sites of these two proteins, which include a pho box and -10 and -35 elements, are proposed.
RESUMEN
Pseudomonas putida strains are ubiquitous in soil and water but have also been reported as opportunistic human pathogens capable of causing nosocomial infections. In this study we describe the multilocus sequence typing of four P. putida strains (HB13667, HB8234, HB4184, and HB3267) isolated from in-patients at the Besançon Hospital (France). The four isolates (in particular HB3267) were resistant to a number of antibiotics. The pathogenicity and virulence potential of the strains was tested ex vivo and in vivo using different biological models: human tissue culture, mammalian tissues, and insect larvae. Our results showed a significant variability in the ability of the four strains to damage the host; HB13667 did not exhibit any pathogenic traits, HB4184 caused damage only ex vivo in human tissue cultures, and HB8234 had a deleterious effect in tissue culture and in vivo on rat skin, but not in insect larvae. Interestingly, strain HB3267 caused damage in all the model systems studied. The putative evolution of these strains in medical environments is discussed.
RESUMEN
The Fox system of Pseudomonas aeruginosa is a cell-surface signaling (CSS) pathway employed by the bacterium to sense and respond to the presence of the heterologous siderophore ferrioxamine in the environment. This regulatory pathway controls the transcription of the foxA ferrioxamine receptor gene through the extracytoplasmic function sigma factor σ(FoxI). In the absence of ferrioxamine, the activity of σ(FoxI) is inhibited by the transmembrane anti-sigma factor FoxR. Upon binding of ferrioxamine by the FoxA receptor, FoxR is processed by a complex proteolytic cascade leading to the release and activation of σ(FoxI). Interestingly, we have recently shown that FoxR undergoes self-cleavage between the periplasmic Gly-191 and Thr-192 residues independent of the perception of ferrioxamine. This autoproteolytic event, which is widespread among CSS anti-sigma factors, produces two distinct domains that interact and function together to transduce the presence of the signal. In this work, we provide evidence that the self-cleavage of FoxR is not an enzyme-dependent process but is induced by an N-O acyl rearrangement. Mutation analysis showed that the nucleophilic side chain of the Thr-192 residue at +1 of the cleavage site is required for an attack on the preceding Gly-191, after which the resulting ester bond is likely hydrolyzed. Because the cleavage site is well preserved and the hydrolysis of periplasmic CSS anti-sigma factors is widely observed, we hypothesize that cleavage via an N-O acyl rearrangement is a conserved feature of these proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Periplasma/metabolismo , Pseudomonas aeruginosa/metabolismo , Receptores de Superficie Celular/metabolismo , Sideróforos/química , Factor sigma/metabolismo , Dominio Catalítico , Análisis Mutacional de ADN , Deferoxamina/química , Escherichia coli/metabolismo , Compuestos Férricos/química , Regulación Bacteriana de la Expresión Génica , Glicina/química , Hidrólisis , Plásmidos/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Treonina/químicaRESUMEN
Cell-surface signalling (CSS) enables Gram-negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti-sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic function (ECF) sigma factor. Recent studies have demonstrated that RseP-mediated proteolysis of the anti-sigma factors is key to σ(ECF) activation. Using the Pseudomonas aeruginosaâ FoxR anti-sigma factor, we show here that RseP is responsible for the generation of an N-terminal tail that likely contains pro-sigma activity. Furthermore, it has been reported previously that this anti-sigma factor is processed in two separate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the CSS signal. However, our results also indicate that this processing event is not essential for activity. In fact, we have identified functional CSS anti-sigma factors that are not cleaved prior to signal perception. Together, our results indicate that CSS regulation can occur through both complete and initially processed anti-sigma factors.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Interacción Gen-Ambiente , Pseudomonas aeruginosa/metabolismo , Receptores de Superficie Celular/metabolismo , Factor sigma/metabolismo , Secuencia de Aminoácidos , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteolisis , Pseudomonas aeruginosa/genética , Transducción de Señal/genéticaRESUMEN
Membrane-spanning signaling pathways enable bacteria to alter gene expression in response to extracytoplasmic stimuli. Many such pathways are cell-surface signaling (CSS) systems, which are tripartite molecular devices that allow Gram-negative bacteria to transduce an extracellular stimulus into a coordinated transcriptional response. Typically, CSS systems are composed of the following: (1) an outer membrane receptor, which senses the extracellular stimulus; (2) a cytoplasmic membrane-spanning protein involved in signal transduction from the periplasm to the cytoplasm; and (3) an extracytoplasmic function (ECF) sigma factor that initiates expression of the stimulus-responsive gene(s). Members of genus Pseudomonas provide a paradigmatic example of how CSS systems contribute to the global control of gene expression. Most CSS systems enable self-regulated uptake of iron via endogenous (pyoverdine) or exogenous (xenosiderophores, heme, and citrate) carriers. Some are also implicated in virulence, biofilm formation, and cell-cell interactions. Incorporating insights from the well-characterized alginate regulatory circuitry, this review will illustrate common themes and variations at the level of structural and functional properties of Pseudomonas CSS systems. Control of the expression and activity of ECF sigma factors are central to gene regulation via CSS, and the variety of intrinsic and extrinsic factors influencing these processes will be discussed.
Asunto(s)
Hierro/metabolismo , Pseudomonas/fisiología , Transducción de Señal , Membrana Celular , Regulación Bacteriana de la Expresión Génica , Pseudomonas/genética , Pseudomonas/metabolismo , Pseudomonas/patogenicidad , Factor sigma/metabolismo , Estrés FisiológicoRESUMEN
In the last years, the zebrafish (Danio rerio) has become an important vertebrate animal model to study host-pathogen interactions, especially in its embryonic stage. The presence of a fully developed innate immune system in the first days of embryogenesis, the facility of obtaining and manipulating large numbers of embryos, the optical transparency of the embryos that allow the direct visualization of bacterial infections, a wide range of genetic tools, and extensive mutant resources and collections of transgenic reporter lines are important advantages of the zebrafish-embryo model. Pseudomonas aeruginosa is able to lethally infect zebrafish embryos when the amount of cells injected exceeds the phagocytic capacity of the embryo. Different studies have proved the suitability of zebrafish embryos as a model to analyze P. aeruginosa infection. Here we describe the detailed protocols to establish a P. aeruginosa infection in zebrafish embryos and to image the interaction of the bacterium with this host with fluorescent microscopy.
Asunto(s)
Bioensayo/métodos , Interacciones Huésped-Patógeno , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Pez Cebra/microbiología , Animales , Embrión no Mamífero/microbiología , Imagenología Tridimensional , Inyecciones , Mamíferos , Virulencia , Pez Cebra/embriologíaRESUMEN
Extracytoplasmic function (ECF) sigma factors play a key role in the regulation of vital functions in the bacterial response to the environment. In Gram-negative bacteria, activity of these sigma factors is often controlled by cell-surface signalling (CSS), a regulatory system that also involves an outer membrane receptor and a transmembrane anti-sigma factor. To get more insight into the molecular mechanism behind CSS regulation, we have focused on the unique Iut system of Pseudomonas putida. This system contains a hybrid protein containing both a cytoplasmic ECF sigma domain and a periplasmic anti-sigma domain, apparently leading to a permanent interaction between the sigma and anti-sigma factor. We show that the Iut ECF sigma factor regulates the response to aerobactin under iron deficiency conditions and is activated by a proteolytic pathway that involves the sequential action of two proteases: Prc, which removes the periplasmic anti-sigma domain, and RseP, which subsequently removes the transmembrane domain and thereby generates the ECF active transcriptional form. We furthermore demonstrate the role of these proteases in the regulation of classical CSS systems in which the sigma and anti-sigma factors are two different proteins.
Asunto(s)
Endopeptidasas/genética , Regulación Bacteriana de la Expresión Génica , Pseudomonas putida/genética , Transducción de Señal/genética , Membrana Celular/metabolismo , Endopeptidasas/metabolismo , Ácidos Hidroxámicos/metabolismo , Hierro/metabolismo , Periplasma/metabolismo , Proteolisis , Pseudomonas putida/metabolismo , Factor sigma/metabolismoRESUMEN
The cell-surface signalling (CSS) system represents an important regulatory mechanism by which Gram-negative bacteria respond to the environment. Gene regulation by CSS systems is particularly present and important in the opportunistic human pathogen Pseudomonas aeruginosa. In this bacterium, these mechanisms regulate mainly the uptake of iron, but also virulence functions. The latter is the case for the P. aeruginosa PUMA3 CSS system formed by the putative VreA receptor, the σ(VreI) extracytoplasmic function sigma factor and the VreR anti-sigma factor. A role for this system in P. aeruginosa virulence has been demonstrated previously. However, the conditions under which this system is expressed and activated have not been elucidated so far. In this work, we have identified and characterized the global regulatory cascade activating the expression of the PUMA3 system. We show that the PhoB transcriptional regulator, part of the PhoB-PhoR two-component signalling system, can sense a limitation of inorganic phosphate to turn on the expression of the vreA, vreI and vreR genes, which constitute an operon. Upon expression of these genes in this condition, σ(VreI) factor mediates transcription of most, but not all, of the previously identified σ(VreI)-regulated genes. Indeed, we found new σ(VreI)-targeted genes and we show that σ(VreI)-regulon genes are all located immediately downstream to the vreAIR gene cluster.