16 research outputs found

    Moonlighting chaperone activity of the enzyme PqsE contributes to RhlR-controlled virulence of Pseudomonas aeruginosa

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    Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa, with the rhl circuit playing the leading role in late and chronic infections. The majority of traits controlled by rhl transcription factor RhlR depend on PqsE, a dispensable thioesterase in Pseudomonas Quinolone Signal (PQS) biosynthesis that interferes with RhlR through an enigmatic mechanism likely involving direct interaction of both proteins. Here we show that PqsE and RhlR form a 2:2 protein complex that, together with RhlR agonist N-butanoyl-L-homoserine lactone (C4-HSL), solubilizes RhlR and thereby renders the otherwise insoluble transcription factor active. We determine crystal structures of the complex and identify residues essential for the interaction. To corroborate the chaperone-like activity of PqsE, we design stability-optimized variants of RhlR that bypass the need for C4-HSL and PqsE in activating PqsE/RhlR-controlled processes of P. aeruginosa. Together, our data provide insight into the unique regulatory role of PqsE and lay groundwork for developing new P. aeruginosa-specific pharmaceuticals

    A global experiment on motivating social distancing during the COVID-19 pandemic

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    Finding communication strategies that effectively motivate social distancing continues to be a global public health priority during the COVID-19 pandemic. This cross-country, preregistered experiment (n = 25,718 from 89 countries) tested hypotheses concerning generalizable positive and negative outcomes of social distancing messages that promoted personal agency and reflective choices (i.e., an autonomy-supportive message) or were restrictive and shaming (i.e., a controlling message) compared with no message at all. Results partially supported experimental hypotheses in that the controlling message increased controlled motivation (a poorly internalized form of motivation relying on shame, guilt, and fear of social consequences) relative to no message. On the other hand, the autonomy-supportive message lowered feelings of defiance compared with the controlling message, but the controlling message did not differ from receiving no message at all. Unexpectedly, messages did not influence autonomous motivation (a highly internalized form of motivation relying on one’s core values) or behavioral intentions. Results supported hypothesized associations between people’s existing autonomous and controlled motivations and self-reported behavioral intentions to engage in social distancing. Controlled motivation was associated with more defiance and less long-term behavioral intention to engage in social distancing, whereas autonomous motivation was associated with less defiance and more short- and long-term intentions to social distance. Overall, this work highlights the potential harm of using shaming and pressuring language in public health communication, with implications for the current and future global health challenges

    DĂ©couverte et caractĂ©risation d’une nouvelle voie d’acquisition des mĂ©taux mĂ©diĂ©e par un metallophore chez Pseudomonas aeruginosa

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    Les mĂ©taux biologiques sont nĂ©cessaires Ă  la croissance et Ă  la survie de tous les organismes vivants. Une stratĂ©gie conservĂ©e chez les bactĂ©ries est la biosynthĂšse de petites molĂ©cules avec une forte affinitĂ© pour les mĂ©taux nommĂ© mĂ©tallophores, leur sĂ©crĂ©tion dans le milieu environnant, et la rĂ©absorption d'un complexe mĂ©tal-mĂ©tallophore par des rĂ©cepteurs spĂ©cifiques.Durant ma thĂšse, j'ai contribuĂ© Ă  la dĂ©couverte d'une nouvelle voie d'import des mĂ©taux mĂ©diĂ©e par un mĂ©tallophore. Les quatre gĂšnes cnt de Pseudomonas aeruginosa sont inclus dans un opĂ©ron, impliquĂ© dans la biosynthĂšse (cntL & cntM), l’export au travers de la membrane interne (MI, cntI) et l’import Ă  travers de la membrane externe (ME, cntO), d'un nouveau mĂ©tallophore que nous avons nommĂ© pseudopaline. (Lhospice*, Gomez*, Ouerdane* et al., Scientific reports, 2017). Nous avons dĂ©terminĂ© que la pseudopaline est sĂ©crĂ©tĂ©e en deux Ă©tapes. La premiĂšre Ă©tape de passage au travers de la MI est assurĂ© par CntI, et la deuxiĂšme Ă©tape de passage au travers de la ME est assurĂ© par la pompe d'efflux MexAB couplĂ©e Ă  OprM (Gomez et al., soumis). Quand le complexe mĂ©tal-pseudopaline est importĂ© dans le pĂ©riplasme aprĂšs avoir Ă©tĂ© reconnu par le rĂ©cepteur CntO, il est modifiĂ© par un mĂ©canisme encore inconnu que nous mettons en Ă©vidence, permettant probablement la libĂ©ration du mĂ©tal. Enfin, nous avons constatĂ© que la machinerie pseudopaline est liĂ©e Ă  l'activitĂ© exoprotease. IMPa, un facteur de virulence avec un effet immunomodulateur, bĂ©nĂ©ficie du Zn acquis par la machinerie pseudopaline (Gomez et al., en prĂ©paration), ce qui constitue une premiĂšre explication au rĂŽle de la pseudopaline durant les phases d’infection.Biological metal are necessary for the growth and survival of all organisms. A shared strategy between bacteria is the biosynthesis of small molecules with an outstanding affinity for metals called metallophores, their secretion in the extracellular space, and the recovery of a metal-complexed metallophore through dedicated membrane transporters.During my PhD, I contributed to the discovery of a new metal uptake pathway mediated by a previously unknown metallophore. The four cnt genes (PA4834-4837) of Pseudomonas aeruginosa are embedded in an operon, involved in the biosynthesis (cntL and cntM), inner membrane (IM) export (cntI) and outer membrane (OM) recovery (cntO) of a nicotianamine-like metallophore that we named pseudopaline (Lhospice*, Gomez*, Ouerdane* et al. 2017). We determined that pseudopaline’s two steps secretion relies on CntI for IM export and the efflux pump MexAB-OprM for the crossing of the OM (Gomez et al, submitted). When retrieved from the extracellular space through CntO, we showed that the loaded metallophore is modified by an unknown mechanism, conceivably allowing metal release. Lastly, we identified an interplay between pseudopaline and exoproteases activity. IMPa, a virulence factor with an immuno-modulator function, directly benefit from zinc acquired by pseudopaline function (Gomez et al, in preparation), thus, constituting a possible explanation to the important role of this metal uptake pathway during infection

    An expanded CRISPR–Cas9-assisted recombineering toolkit for engineering genetically intractable<i> Pseudomonas aeruginosa</i> isolates

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    Much of our current understanding of microbiology is based on the application of genetic engineering procedures. Since their inception (more than 30 years ago), methods based largely on allelic exchange and two-step selection processes have become a cornerstone of contemporary bacterial genetics. While these tools are established for adapted laboratory strains, they have limited applicability in clinical or environmental isolates displaying a large and unknown genetic repertoire that are recalcitrant to genetic modifications. Hence, new tools allowing genetic engineering of intractable bacteria must be developed to gain a comprehensive understanding of them in the context of their biological niche. Herein, we present a method for precise, efficient and rapid engineering of the opportunistic pathogen Pseudomonas aeruginosa. This procedure relies on recombination of short single-stranded DNA facilitated by targeted double-strand DNA breaks mediated by a synthetic Cas9 coupled with the efficient Ssr recombinase. Possible applications include introducing single-nucleotide polymorphisms, short or long deletions, and short DNA insertions using synthetic single-stranded DNA templates, drastically reducing the need of PCR and cloning steps. Our toolkit is encoded on two plasmids, harboring an array of different antibiotic resistance cassettes; hence, this approach can be successfully applied to isolates displaying natural antibiotic resistances. Overall, this toolkit substantially reduces the time required to introduce a range of genetic manipulations to a minimum of five experimental days, and enables a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates

    Involvement of the Pseudomonas aeruginosa MexAB–OprM efflux pump in the secretion of the metallophore pseudopaline

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    International audienceTo overcome the metal restriction imposed by the host’s nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore-mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad-spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram-negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB–OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal-loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginos

    Pseudomonas aeruginosa zinc uptake in chelating environment is primarily mediated by the metallophore pseudopaline

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    Abstract Metal uptake is vital for all living organisms. In metal scarce conditions a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here we characterize a four-gene operon (PA4837–PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated with an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The pseudopaline system is involved in nickel uptake in poor media, and, most importantly, in zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as the main zinc importer under these metal scarce conditions

    Procesamiento de imĂĄgenes

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    El semillero tiene como uno de sus objetivos, la enseñanza y la aplicación de técnicas y herramientas de inteligencia artificial en åreas de la ingeniería electromecånica y afines. Para ello se seguirå un proceso que requerirå en sus primeras etapas la recopilación de la información, su limpieza, transformación y anålisis, persiguiendo mediante el aprendizaje continuo de los estudiantes y su desarrollo en posteriores etapas, la implementación de modelos y/o arquitecturas que permitan desarrollar un modelo de IA basado en técnicas de visión por computadora y aprendizaje automåtico para reconocer las placas de los vehículos que ingresan a la ETITC en tiempo real y/o aplicaciones en general, como procesos de regresión, clasificación, segmentación, etc. Considerando que a futuro se plantearå el trabajar con imågenes, se sabe que este campo presenta gran auge en distintos campos, pues como lo menciona LeCun et al. 2015, el uso de redes convolucionales ha ampliado la capacidad para extraer características relevantes de las imågenes, lo que es fundamental para el reconocimiento de placas de vehículos. Adicionalmente, se han desarrollado métodos como el introducido por Redmon J et al. (2016), el cual es conocido actualmente como YOLO "You Only Look Once" que mediante redes convolucionales facilita el reconocimiento de objetos. Adicionalmente se tiene el ejemplo de Krizhevsky, A (2012), quien mediante el modelo AlexNet, presentó gran eficacia en tareas de reconocimiento de imågenes

    Author Correction: A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

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    Correction to: Nature Human Behaviour https://doi.org/10.1038/s41562-021-01173-x, published online 2 August 2021. In the version of this article initially published, the following authors were omitted from the author list and the Author contributionssection for “investigation” and “writing and editing”: Nandor Hajdu (Institute of Psychology, ELTE Eötvös LorĂĄnd University, Budapest,Hungary), Jordane Boudesseul (Facultad de PsicologĂ­a, Instituto de InvestigaciĂłn CientĂ­fica, Universidad de Lima, Lima, PerĂș), RafaƂMuda (Faculty of Economics, Maria Curie-Sklodowska University, Lublin, Poland) and Sandersan Onie (Black Dog Institute, UNSWSydney, Sydney, Australia & Emotional Health for All Foundation, Jakarta, Indonesia). In addition, Saeideh FatahModares’ name wasoriginally misspelled as Saiedeh FatahModarres in the author list. Further, affiliations have been corrected for Maria Terskova (NationalResearch University Higher School of Economics, Moscow, Russia), Susana Ruiz Fernandez (FOM University of Applied Sciences,Essen; Leibniz-Institut fur Wissensmedien, Tubingen, and LEAD Research Network, Eberhard Karls University, Tubingen, Germany),Hendrik Godbersen (FOM University of Applied Sciences, Essen, Germany), Gulnaz Anjum (Department of Psychology, Simon FraserUniversity, Burnaby, Canada, and Department of Economics & Social Sciences, Institute of Business Administration, Karachi, Pakistan)

    Author correction: A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic

    No full text
    Correction to: Nature Human Behaviour https://doi.org/10.1038/s41562-021-01173-x, published online 2 August 2021. In the version of this article initially published, the following authors were omitted from the author list and the Author contributionssection for “investigation” and “writing and editing”: Nandor Hajdu (Institute of Psychology, ELTE Eötvös LorĂĄnd University, Budapest,Hungary), Jordane Boudesseul (Facultad de PsicologĂ­a, Instituto de InvestigaciĂłn CientĂ­fica, Universidad de Lima, Lima, PerĂș), RafaƂMuda (Faculty of Economics, Maria Curie-Sklodowska University, Lublin, Poland) and Sandersan Onie (Black Dog Institute, UNSWSydney, Sydney, Australia & Emotional Health for All Foundation, Jakarta, Indonesia). In addition, Saeideh FatahModares’ name wasoriginally misspelled as Saiedeh FatahModarres in the author list. Further, affiliations have been corrected for Maria Terskova (NationalResearch University Higher School of Economics, Moscow, Russia), Susana Ruiz Fernandez (FOM University of Applied Sciences,Essen; Leibniz-Institut fur Wissensmedien, Tubingen, and LEAD Research Network, Eberhard Karls University, Tubingen, Germany),Hendrik Godbersen (FOM University of Applied Sciences, Essen, Germany), Gulnaz Anjum (Department of Psychology, Simon FraserUniversity, Burnaby, Canada, and Department of Economics & Social Sciences, Institute of Business Administration, Karachi, Pakistan)
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