Chemical communication between synthetic and natural cells: a possible experimental design

The bottom-up construction of synthetic cells is one of the most intriguing and interesting research arenas in synthetic biology. Synthetic cells are built by encapsulating biomolecules inside lipid vesicles (liposomes), allowing the synthesis of one or more functional proteins. Thanks to the in situ synthesized proteins, synthetic cells become able to perform several biomolecular functions, which can be exploited for a large variety of applications. This paves the way to several advanced uses of synthetic cells in basic science and biotechnology, thanks to their versatility, modularity, biocompatibility, and programmability. In the previous WIVACE (2012) we presented the state-of-the-art of semi-synthetic minimal cell (SSMC) technology and introduced, for the first time, the idea of chemical communication between synthetic cells and natural cells. The development of a proper synthetic communication protocol should be seen as a tool for the nascent field of bio/chemical-based Information and Communication Technologies (bio-chem-ICTs) and ultimately aimed at building soft-wet-micro-robots. In this contribution (WIVACE, 2013) we present a blueprint for realizing this project, and show some preliminary experimental results. We firstly discuss how our research goal (based on the natural capabilities of biological systems to manipulate chemical signals) finds a proper place in the current scientific and technological contexts. Then, we shortly comment on the experimental approaches from the viewpoints of (i) synthetic cell construction, and (ii) bioengineering of microorganisms, providing up-to-date results from our laboratory. Finally, we shortly discuss how autopoiesis can be used as a theoretical framework for defining synthetic minimal life, minimal cognition, and as bridge between synthetic biology and artificial intelligence.Comment: In Proceedings Wivace 2013, arXiv:1309.712

The interplay of StyR and IHF regulates substrate-dependent induction and carbon catabolite repression of styrene catabolism genes in Pseudomonas fluorescens ST

<p>Abstract</p> <p>Background</p> <p>In <it>Pseudomonas fluorescens </it>ST, the promoter of the styrene catabolic operon, P<it>styA</it>, is induced by styrene and is subject to catabolite repression. P<it>styA </it>regulation relies on the StyS/StyR two-component system and on the IHF global regulator. The phosphorylated response regulator StyR (StyR-P) activates P<it>styA </it>in inducing conditions when it binds to the high-affinity site STY2, located about -40 bp from the transcription start point. A <it>cis</it>-acting element upstream of STY2, named URE, contains a low-affinity StyR-P binding site (STY1), overlapping the IHF binding site. Deletion of the URE led to a decrease of promoter activity in inducing conditions and to a partial release of catabolite repression. This study was undertaken to assess the relative role played by IHF and StyR-P on the URE, and to clarify if P<it>styA </it>catabolite repression could rely on the interplay of these regulators.</p> <p>Results</p> <p>StyR-P and IHF compete for binding to the URE region. P<it>styA </it>full activity in inducing conditions is achieved when StyR-P and IHF bind to site STY2 and to the URE, respectively. Under catabolite repression conditions, StyR-P binds the STY1 site, replacing IHF at the URE region. StyR-P bound to both STY1 and STY2 sites oligomerizes, likely promoting the formation of a DNA loop that closes the promoter in a repressed conformation. We found that StyR and IHF protein levels did not change in catabolite repression conditions, implying that P<it>styA </it>repression is achieved through an increase in the StyR-P/StyR ratio.</p> <p>Conclusion</p> <p>We propose a model according to which the activity of the P<it>styA </it>promoter is determined by conformational changes. An open conformation is operative in inducing conditions when StyR-P is bound to STY2 site and IHF to the URE. Under catabolite repression conditions StyR-P cellular levels would increase, displacing IHF from the URE and closing the promoter in a repressed conformation. The balance between the open and the closed promoter conformation would determine a fine modulation of the promoter activity. Since StyR and IHF protein levels do not vary in the different conditions, the key-factor regulating P<it>styA </it>catabolite repression is likely the kinase activity of the StyR-cognate sensor protein StyS.</p

Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and virulence

Efflux pumps of the resistance-nodulation-cell-division (RND) family increase antibiotic resistance in many bacterial pathogens, representing candidate targets for the development of antibiotic adjuvants. RND pumps have also been proposed to contribute to bacterial infection, implying that efflux pump inhibitors (EPIs) could also act as anti-virulence drugs. Nevertheless, EPIs are usually investigated only for their properties as antibiotic adjuvants, while their potential anti-virulence activity is seldom taken into account. In this study it is shown that RND efflux pumps contribute to Pseudomonas aeruginosa PAO1 pathogenicity in an insect model of infection, and that the well-characterized EPI Phe-Arg-β-naphthylamide (PAβN) is able to reduce in vivo virulence of the P. aeruginosa PAO1 laboratory strain, as well as of clinical isolates. The production of quorum sensing (QS) molecules and of QS-dependent virulence phenotypes is differentially affected by PAβN, depending on the strain. Transcriptomic and phenotypic analyses showed that the protection exerted by PAβN from P. aeruginosa PAO1 infection in vivo correlates with the down-regulation of key virulence genes (e.g. genes involved in iron and phosphate starvation). Since PAβN impacts P. aeruginosa virulence, anti-virulence properties of EPIs are worthy to be explored, taking into account possible strain-specificity of their activit

Identification of FDA-Approved Drugs as Antivirulence Agents Targeting the pqs Quorum-Sensing System of Pseudomonas aeruginosa

Copyright © 2018 American Society for Microbiology. All Rights Reserved. The long-term use of antibiotics has led to the emergence of multidrug-resistant bacteria. A promising strategy to combat bacterial infections aims at hampering their adaptability to the host environment without affecting growth. In this context, the intercellular communication system quorum sensing (QS), which controls virulence factor production and biofilm formation in diverse human pathogens, is considered an ideal target. Here, we describe the identification of new inhibitors of the pqs QS system of the human pathogen Pseudomonas aeruginosa by screening a library of 1,600 U.S. Food and Drug Administration-approved drugs. Phenotypic characterization of ad hoc engineered strains and in silico molecular docking demonstrated that the antifungal drugs clotrimazole and miconazole, as well as an antibacterial compound active against Gram-positive pathogens, clofoctol, inhibit the pqs system, probably by targeting the transcriptional regulator PqsR. The most active inhibitor, clofoctol, specifically inhibited the expression of pqs-controlled virulence traits in P. aeruginosa, such as pyocyanin production, swarming motility, biofilm formation, and expression of genes involved in siderophore production. Moreover, clofoctol protected Galleria mellonella larvae from P. aeruginosa infection and inhibited the pqs QS system in P. aeruginosa isolates from cystic fibrosis patients. Notably, clofoctol is already approved for clinical treatment of pulmonary infections caused by Gram-positive bacterial pathogens; hence, this drug has considerable clinical potential as an antivirulence agent for the treatment of P. aeruginosa lung infections

Alkyl-quinolone-dependent quorum sensing controls prophage-mediated autolysis in Pseudomonas aeruginosa colony biofilms

Pseudomonas aeruginosa is a model quorum sensing (QS) pathogen with three interconnected QS circuits that control the production of virulence factors and antibiotic tolerant biofilms. The pqs QS system of P. aeruginosa is responsible for the biosynthesis of diverse 2-alkyl-4-quinolones (AQs), of which 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) function as QS signal molecules. Transcriptomic analyses revealed that HHQ and PQS influenced the expression of multiple genes via PqsR-dependent and -independent pathways whereas 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) had no effect on P. aeruginosa transcriptome. HQNO is a cytochrome bc 1 inhibitor that causes P. aeruginosa programmed cell death and autolysis. However, P. aeruginosa pqsL mutants unable to synthesize HQNO undergo autolysis when grown as colony biofilms. The mechanism by which such autolysis occurs is not understood. Through the generation and phenotypic characterization of multiple P. aeruginosa PAO1 mutants producing altered levels of AQs in different combinations, we demonstrate that mutation of pqsL results in the accumulation of HHQ which in turn leads to Pf4 prophage activation and consequently autolysis. Notably, the effect of HHQ on Pf4 activation is not mediated via its cognate receptor PqsR. These data indicate that the synthesis of HQNO in PAO1 limits HHQ-induced autolysis mediated by Pf4 in colony biofilms. A similar phenomenon is shown to occur in P. aeruginosa cystic fibrosis (CF) isolates, in which the autolytic phenotype can be abrogated by ectopic expression of pqsL

RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing

In its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that considerable cell-to-cell variation in the QS activation state occurs during growth, often resulting in coexisting subpopulations of cells in which QS is active (quorate cells) or inactive (non-quorate cells). Heterogeneity has been observed in the las QS system of the opportunistic pathogen Pseudomonas aeruginosa. However, the molecular mechanisms underlying this phenomenon have not yet been defined. The las QS system consists of an incoherent feedforward loop in which the LasR transcriptional regulator activates the expression of the lasI synthase gene and rsaL, coding for the lasI transcriptional repressor RsaL. Here, single-cell-level gene expression analyses performed in ad hoc engineered biosensor strains and deletion mutants revealed that direct binding of RsaL to the lasI promoter region increases heterogeneous activation of the las QS system. Experiments performed with a dual-fluorescence reporter system showed that the LasR-dependent expression of lasI and rsaL does not correlate in single cells, indicating that RsaL acts as a brake that stochastically limits the transition of non-quorate cells to the quorate state in a subpopulation of cells expressing high levels of this negative regulator. Interestingly, the rhl QS system that is not controlled by an analogous RsaL protein showed higher homogeneity with respect to the las system

IAU Office of Astronomy for Education, OAE Center Italy - Annual Report 2022

Annual report 2023 of the the IAU OAE Center Italy, an international office addressed to education and hosted and financed by Inaf. OAE Center Italy was established on the 3rd of March 2021, thanks to a Memorandum of Understanding signed by three parties: IAU, the Office of Astronomy for Education and INAF. OAE Center Italy is a joint project of a consortium of Italian partners, led and represented by INAF and of the IAU OAE, and is operated by INAF. The Italian partners are INAF, the Italian Astronomical Society (SAIt) and the University of Rome Tor Vergata (ToV). Index of contents 1. Professionalize astronomy education 11 1.1 The 4th Shaw-IAU workshop of Astronomy for Education 12 1.2 “Officina degli Errori” Pilot Programme 14 1.3 Educational path for transversal skills and professional orientation through Game-Based Learning 16 2. Provide access to good resources 18 2.1 astroEDU 19 2.2 Shared astronomical Glossary 21 2.3 Astrophotography contest 23 3. Promote astronomy in curricula 24 3.1 MIRTO - Mediterranean Informal Round Table – online 25 3.2 STEAM-MED - Lampedusa workshop on Astronomy Education 2022 27 3.3 Teacher Training Pilot Program (TTPP) 35 3.4 Organisation and delivery of the 16 hour-course for PhD students "Designing innovative public-engagement activities", at the University of Padova. 36 3.5 Castellaro Lagusello Astronomy Festival 37 4. OAE Networking 38 4.1 Participation in Expo 2020, Dubai, Italian Pavilion 39 4.2 Under the same Moon: building bridges through sky and astronomy 40 5. Spread the news 41 5.1 Under the same sky: an exhibition in Rome 42 5.2 Il Cielo itinerante – Italy shines 42 5.3 Universe World / EduINAF column 43 5.4 Time machines, an astronomy exhibition 43 5.5 Workshops 43 5.6 Press release, announcements and new

IAU Office of Astronomy for Education, OAE Center Italy - Annual Report 2021

First annual report of the the IAU OAE Center Italy, an international office addressed to education and hosted and financed by Inaf. OAE Center Italy was established on the 3rd of March 2021, thanks to a Memorandum of Understanding signed by three parties: IAU, the Office of Astronomy for Education and INAF. OAE Center Italy is a joint project of a consortium of Italian partners, led and represented by INAF and of the IAU OAE, and is operated by INAF. The Italian partners are INAF, the Italian Astronomical Society (SAIt) and the University of Rome Tor Vergata (ToV)

IAU Office of Astronomy for Education OAE Center Italy - Quarterly Report 1 January-March 2022

The IAU O!ce of Astronomy for Education Center Italy (I-OAE) is a joint project of a consortium of Italian partners led and represented by Istituto Nazionale di Astrofisica (INAF, National Institute for Astrophysics), the International Astronomical Union (IAU) and the IAU O!ce of Astronomy for Education. The Italian consortium is constituted by: INAF, the Italian Astronomical Society (SAIt) and the University of Rome Tor Vergata (ToV). I-OAE HQ are hosted by the INAF - Rome Astronomical Observatory, in Monteporzio. Personnel is selected on voluntary bases according to their interests and competence, in agreement with the Institutes they work for. Brochure of the 2021 activities: https://www.flipsnack.com/eduinaf/oaei-brochure.htmlQuarterly Report 1 January-March 2022 is the report of the activities from January to March 2022 of the IAU Office of Astronomy for Education OAE Center Italy Attachment: brochure of the activities and project 202

The antibacterial activity of acetic acid against biofilm-producing pathogens of relevance to burns patients

Introduction: Localised infections, and burn wound sepsis are key concerns in the treatment of burns patients, and prevention of colonisation largely relies on biocides. Acetic acid has been shown to have good antibacterial activity against various planktonic organisms, however data is limited on efficacy, and few studies have been performed on biofilms. Objectives: We sought to investigate the antibacterial activity of acetic acid against important burn wound colonising organisms growing planktonically and as biofilms. Methods: Laboratory experiments were performed to test the ability of acetic acid to inhibit growth of pathogens, inhibit the formation of biofilms, and eradicate pre-formed biofilms. Results: Twenty-nine isolates of common wound-infecting pathogens were tested. Acetic acid was antibacterial against planktonic growth, with an minimum inhibitory concentration of 0.16-0.31% for all isolates, and was also able to prevent formation of biofilms (at 0.31 %). Eradication of mature biofilms was observed for all isolates after three hours of exposure. Conclusions: This study provides evidence that acetic acid can inhibit growth of key burn wound pathogens when used at very dilute concentrations. Owing to current concerns of the reducing efficacy of systemic antibiotics, this novel biocide application offers great promise as a cheap and effective measure to treat infections in burns patients