Revisiting regulation of potassium homeostasis in Escherichia coli:the connection to phosphate limitation

Two-component signal transduction constitutes the predominant strategy used by bacteria to adapt to fluctuating environments. The KdpD/KdpE system is one of the most widespread, and is crucial for K+ homeostasis. In Escherichia coli, the histidine kinase KdpD senses K+ availability, whereas the response regulator KdpE activates synthesis of the high-affinity K+ uptake system KdpFABC. Here we show that, in the absence of KdpD, kdpFABC expression can be activated via phosphorylation of KdpE by the histidine kinase PhoR. PhoR and its cognate response regulator PhoB comprise a phosphate-responsive two-component system, which senses phosphate limitation indirectly through the phosphate transporter PstCAB and its accessory protein PhoU. In vivo two-hybrid interaction studies based on the bacterial adenylate cyclase reveal pairwise interactions between KdpD, PhoR, and PhoU. Finally, we demonstrate that cross-regulation between the kdpFABC and pstSCAB operons occurs in both directions under simultaneous K+ and phosphate limitation, both in vitro and in vivo. This study for the first time demonstrates direct coupling between intracellular K+ and phosphate homeostasis and provides a mechanism for fine-tuning of the balance between positively and negatively charged ions in the bacterial cell

Equation-Free Analysis of Two-Component System Signalling Model Reveals the Emergence of Co-Existing Phenotypes in the Absence of Multistationarity

Phenotypic differences of genetically identical cells under the same environmental conditions have been attributed to the inherent stochasticity of biochemical processes. Various mechanisms have been suggested, including the existence of alternative steady states in regulatory networks that are reached by means of stochastic fluctuations, long transient excursions from a stable state to an unstable excited state, and the switching on and off of a reaction network according to the availability of a constituent chemical species. Here we analyse a detailed stochastic kinetic model of two-component system signalling in bacteria, and show that alternative phenotypes emerge in the absence of these features. We perform a bifurcation analysis of deterministic reaction rate equations derived from the model, and find that they cannot reproduce the whole range of qualitative responses to external signals demonstrated by direct stochastic simulations. In particular, the mixed mode, where stochastic switching and a graded response are seen simultaneously, is absent. However, probabilistic and equation-free analyses of the stochastic model that calculate stationary states for the mean of an ensemble of stochastic trajectories reveal that slow transcription of either response regulator or histidine kinase leads to the coexistence of an approximate basal solution and a graded response that combine to produce the mixed mode, thus establishing its essential stochastic nature. The same techniques also show that stochasticity results in the observation of an all-or-none bistable response over a much wider range of external signals than would be expected on deterministic grounds. Thus we demonstrate the application of numerical equation-free methods to a detailed biochemical reaction network model, and show that it can provide new insight into the role of stochasticity in the emergence of phenotypic diversity

Stochastic activation of the response regulator PhoB by noncognate histidine kinases

Two-component systems (TCS) are the most prevalent gene regulatory mechanism in bacteria. A typical TCS is comprised of a histidine kinase (HK) and a partner response regulator (RR). Specific environment signals lead to autophosphorylation of different HKs, which in turn act as phosphoryl donors for autophosphorylation of their partner RRs. Nonpartner HKs and RRs also interact, giving rise to cross regulation among TCSs in response to diverse signals

Autophosphorylation of the Response Regulator PhoB

Two-component signal transduction systems are prevalent signal transduction systems in microorganisms. The essential elements of two-component systems are the sensor kinase and the response regulator. Response regulators typically receive a phosphoryl group in vivo from a sensor kinase, leading to a conformational change in the response regulator and a downstream effect. However, response regulators can also catalyze their own autophosphorylation with small-molecule phosphodonors. Using autophosphorylation to probe interactions between response regulators and small molecules might give insights potentially applicable to development of small molecule antibiotics. Prior to the work presented here, CheY was the only response regulator for which extensively characterized autophosphorylation kinetics had been published. The relationship between accumulation of phosphorylated CheY and small-molecule phosphodonor concentration remains linear up to the highest concentrations of phosphodonor tested, indicating very weak substrate binding. Here we describe extensive kinetic characterization of autophosphorylation of the Escherichia coli response regulator PhoB. Autophosphorylation kinetics differed greatly between PhoB and CheY. Specifically, the apparent rate constant for accumulation of PhoB-P appeared sigmoidal (Hill coefficient ~2) with respect to small-molecule phosphodonor concentration and to approach saturation. The data are consistent with a model in which PhoB-P forms a heterodimer with an unphosphorylated PhoB monomer, which then autophosphorylates at an enhanced rate. Potential physiological implications of PhoB heterodimers are discussed. Substitutions of nonconserved residues in the PhoB active site appeared to change the autophosphorylation reaction rate but not substrate binding. Further investigation of the link between PhoB dimerization and autophosphorylation using biolayer interferometry is proposed.Doctor of Philosoph

Étude du rôle spécifique du système à deux composantes PhoBR et du système Pst (phosphate specific transport) dans la virulence d’une souche pathogène aviaire de Escherichia coli (APEC)

Les souches d’Escherichia coli pathogènes aviaires (APEC) sont responsables d’infections respiratoires et de septicémies chez la volaille. Le régulon Pho est contrôlé conjointement par le système à deux composantes PhoBR et par le système de transport spécifique du phosphate (Pst). Afin de déterminer l’implication de PhoBR et du système Pst dans la pathogenèse de la souche APEC O78 χ7122, différentes souche mutantes phoBR et pst ont été testées pour divers traits de virulence in vivo et in vitro. Les mutations menant à l’activation constitutive du régulon Pho rendaient les souches plus sensibles au peroxyde d’hydrogène et au sérum de lapin comparativement à la souche sauvage. De plus, l’expression des fimbriae de type 1 était affectée chez ces souches. L’ensemble des mutants Pho-constitutifs étaient aussi significativement moins virulents que la souche sauvage dans un modèle de coinfection de poulet, incluant les souches avec un système Pst fonctionnel. De plus, l’inactivation du régulateur PhoB chez un mutant Pst restaure la virulence. Par ailleurs, l’inactivation de PhoB n’affecte pas la virulence de la souche χ7122 dans notre modèle. De manière intéressante, le degré d’atténuation des souches mutantes corrèle directement avec le niveau d’activation du régulon Pho. Globalement, les résultats indiquent que l’activation du régulon Pho plutôt que le transport du phosphate via le système Pst joue un rôle majeur dans l’atténuation des APEC.Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections and septicemia in poultry. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the involvement of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested for in vivo and in vitro virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild-type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were out-competed by the wild-type parent, including strains containing a functional Pst system. In addition, cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. Loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of APEC

Interaction d'Escherichia coli entérohémorragique (EHEC) avec Acanthamoeba castellanii et rôle du régulon Pho chez les EHEC

Les EHEC de sérotype O157:H7 sont des agents zoonotiques d’origine alimentaire ou hydrique. Ce sont des pathogènes émergeants qui causent chez l’humain des épidémies de gastro-entérite aiguë et parfois un syndrome hémolytique-urémique. Les EHEC réussissent leur transmission à l’humain à partir de leur portage commensal chez l’animal en passant par l’étape de survie dans l’environnement. L’endosymbiose microbienne est une des stratégies utilisées par les bactéries pathogènes pour survivre dans les environnements aquatiques. Les amibes sont des protozoaires vivants dans divers écosystèmes et connus pour abriter plusieurs agents pathogènes. Ainsi, les amibes contribueraient à transmettre les EHEC à l'humain. La première partie de mon projet de thèse est centrée sur l'interaction de l’amibe Acanthamoeba castellanii avec les EHEC. Les résultats montrent que la présence de cette amibe prolonge la persistance des EHEC, et ces dernières survivent à leur phagocytose par les amibes. Ces résultats démontrent le potentiel réel des amibes à héberger les EHEC et à contribuer à leur transmission. Cependant, l’absence de Shiga toxines améliore leur taux de survie intra-amibe. Par ailleurs, les Shiga toxines sont partiellement responsables de l’intoxication des amibes par les EHEC. Cette implication des Shiga toxines dans le taux de survie intracellulaire et dans la mortalité des amibes démontre l’intérêt d’utiliser les amibes comme modèle d'interaction hôte/pathogène pour étudier la pathogénicité des EHEC. Durant leur cycle de transmission, les EHEC rencontrent des carences en phosphate inorganique (Pi) dans l’environnement. En utilisant conjointement le système à deux composantes (TCS) PhoB-R et le système Pst (transport spécifique de Pi), les EHEC détectent et répondent à cette variation en Pi en activant le régulon Pho. La relation entre la virulence des EHEC, le PhoB-R-Pst et/ou le Pi environnemental demeure inconnue. La seconde partie de mon projet explore le rôle du régulon Pho (répondant à un stress nutritif de limitation en Pi) dans la virulence des EHEC. L’analyse transcriptomique montre que les EHEC répondent à la carence de Pi par une réaction complexe impliquant non seulement un remodelage du métabolisme général, qui est critique pour sa survie, mais aussi en coordonnant sa réponse de virulence. Dans ces conditions le régulateur PhoB contrôle directement l’expression des gènes du LEE et de l’opéron stx2AB. Ceci est confirmé par l’augmentation de la sécrétion de l’effecteur EspB et de la production et sécrétion de Stx2 en carence en Pi. Par ailleurs, l’activation du régulon Pho augmente la formation de biofilm et réduit la motilité chez les EHEC. Ceci corrèle avec l’induction des gènes régulant la production de curli et la répression de la voie de production d’indole et de biosynthèse du flagelle et du PGA (Polymère β-1,6-N-acétyle-D-glucosamine).EHEC O157:H7 are an emerging zoonotic food- and water-borne hazard highly pathogenic to humans and associated with diseases ranging from acute gastroenteritis to hemolytic uremic syndrome. From their commensal carriage by farm animals to human targets, EHEC pass through a crucial step of persistence in the open environment. Microbial endosymbiosis is one strategy used by pathogenic bacteria to survive in aquatic environments. Amoebae species are free-living protozoa found in diverse environmental habitats and known to host several water-borne pathogens. Thus amoebae could contribute to transmission of EHEC to humans. The first part of my PhD project was focused on interaction of the free-living amoebae Acanthamoeba castellanii with EHEC. The results showed that the presence of amoeba extends the persistence of EHEC that survived phagocytosis by amoebae. This demonstrates the real potential of amoebae to harbourd EHEC that may contribute to their transmission. However, absence of shiga toxins enhanced the intra-amoeba survival. Moreover, EHEC had a toxic and lethal effect on amoebae partially due to shiga toxins. The involvement of shiga toxins in the intracellular survival and mortality of amoebae suggests the value of using amoebae as a model of host/pathogen interactions to study the pathogenicity of EHEC. During their transmission cycle, EHEC encounter limitation inorganic phosphate (Pi) in the environment. Using jointly the PhoB-R two-component system (TCS) and the Pst (Pi specific transport) system, EHEC detect and respond to this Pi limitation by activating the Pho regulon. The interplay between the EHEC virulence, the Pho-Pst and/or the environmental Pi remains unknown. The second part of my project explored the role of Pho regulon (responding to Pi-limitation stress) in the virulence of EHEC. Transcriptomic analysis showed that EHEC has evolved a sophisticated response to Pi deficiency involving not only biochemical strategies that are likely critical to its survival, but also coordinating its virulence response. In these conditions, the regulator PhoB regulates directly the expression of LEE and Stx2 genes. This is confirmed by an increase in EspB secretion and Stx2 production and secretion in low Pi conditions. Moreover, the activation of Pho regulon increases biofilm formation and reduces motility in EHEC. This correlated with the induction of genes regulating curli production and repression of indole production pathway and the flagellum and PGA biosynthesis