1,721,061 research outputs found
Regolazione del “Quorum Sensing” ed espressione della virulenza in Pseudomonas aeruginosa.
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Molecular Communications in the Context of “Synthetic Cells” Research
No full textThe recent progresses in bottom-up synthetic biology allow the construction of cell-like systems (shortly called “synthetic cells”) based on the encapsulation of chemicals and biological macromolecules inside lipid vesicles. Synthetic cells are far from being alive, but can be designed in order to imitate biological cells with respect to specific functions. The exchange of chemical signals is one of the most fascinating ones. Experimental papers have shown that synthetic cells can be designed to send and receive molecular signals, inaugurating a new research avenue that can be highly relevant not only for nano-medicine and nano-biotechnology, but also for basic understanding of minimal cognitive systems. Here we shortly present the synthetic cell technology and illustrate how to implement the concept of molecular communication in this field
The art of antibacterial warfare: Deception through interference with quorum sensing-mediated communication
No full textAlmost a century on from the discovery of penicillin, the war against bacterial infection still rages compounded by the emergence of strains resistant to virtually every clinically approved antibiotic and the dearth of new antibacterial agents entering the clinic. Consequently there is renewed interest in drugs which attenuate virulence rather than bacterial growth. Since the metaphors of warfare are often used to describe the battle between pathogen and host, we will describe in such a context, the molecular communication (quorum sensing) mechanisms used by bacteria to co-ordinate virulence at the population level. Recent progress in exploiting this information through the design of anti-virulence deception strategies that disrupt quorum sensing through signal molecule inactivation, inhibition of signal molecule biosynthesis or the blockade of signal transduction and their advantages and disadvantages are considered
Development and validation of a novel screening system for the identification of Pseudomonas aeruginosa virulence inhibitor
No full textThe ubiquitous Gram-negative bacterium Pseudomonas aeruginosa is the main agent of lung function decline and mortality in Cystic Fibrosis (CF) patients.Traditional antimicrobial approaches targeting the bacterial viability and growth have proven to be unsuccessful in the treatment of chronic P. aeruginosa infection because of the emergence of resistance. A novel and promising approach is screening for molecules that inhibit the traits that are specifically required for P. aeruginosa virulence.Bacteria live in communities within the infected human host, and their group-behaviour has a deep influence on the expression of virulence traits. The quorum sensing and pyoverdine signalling regulatory systems control the group-behaviour of P. aeruginosa and the expression of virulence factors, biofilm formation and antibiotic resistance, ultimately enhancing the fitness of P. aeruginosa in the host environment.The underlying rationale of this project is the identification of a new generation of drugs acting against the quorum sensing and pyoverdine signalling systems, and hence against the P. aeruginosa ability to cause infection. The use of these novel drugs would provide the host immune system with a better chance of clearing the infection, and could circumvent the problem of the emergence of drug-resistant strains.The specific goal of the project is to set-up, validate and use novel high-throughput screening systems to detect molecules suppressing P. aeruginosa quorum sensing and pyoverdine signalling. The screening systems developed in this project will be used to screen libraries of synthetic and natural compounds. Moreover, these systems will provide the research community with a valuable tool for the discovery of new anti-P. aeruginosa compounds
Iron uptake and quorum sensing in Pseudomonas aeruginosa virulence
No full textPseudomonas aeruginosa is the main cause of lung function decline among cystic fibrosis (CF) patients. The abundance of regulatory genes in the P. aeruginosa genome is at the basis of the environmental versatility of this bacterium, which is mirrored by its ability to efficiently colonise the CF lung. The CF lung is particularly poor in bio-available iron, and iron depletion is sensed by P. aeruginosa as a primary environmental signal that leads to the expression of virulence genes in the host. P. aeruginosa persists in the CF lung thanks to the expression of iron-uptake systems which are themselves regarded to as virulence factors.Establishment of the P. aeruginosa chronic infection requires the development of a microbial community which evolves into a biofilm. In this microbial community the response of each cell to the environmental stimuli originating from the CF lung is coordinated with that of the other cells. The group-behaviour of P. aeruginosa is primarily controlled by the quorum sensing (QS) system that controls the expression of several virulence genes.The first year of the research project has been aimed at identifying and characterizing key molecular factors involved in iron uptake and QS, in order to identify suitable targets for new-drugs development.We have i characterized the CysB protein, a novel transcription factor involved in the regulation of iron uptake and, likely, virulence genes,. Moreover, by mens of a proteomic approach, we have identified about 200 proteins present in P. aeruginosa periplasm, including factors likely involved in iron uptake.Concerning QS regulation, we have showed, by transcriptome analysis, that the RsaL protein is a global regulator controlling hundreds of P. aeruginosa genes, including QS genes and genes involved in virulence. Functional studies have showed that RsaL on one hand plays a positive role in biofilm production and on the other hand maintains the production of important virulence factors within profitable limits. These results suggest that RsaL could be important in the chronic CF lung infection, characterized by biofilm formation and low level of virulence factors production.In the second year of the project we will continue the characterization of the above factors and their contribution to the lung infection. In order to identify new putative drug targets, we will also study the interplay between QS and iron uptake circuits
Biosensor for N-3-oxo-dodecanoyl-homoserin lactone detection
No full textLa presente invenzione concerne la costruzione di un biosensore per la rilevazione di
3OC12-HSL, il principale segnale di quorum sensing (QS) prodotto da Pseudomonas
aeruginosa, in campioni di laboratorio ed in campioni clinici, e per lo screening di inibitori
del sistema 3OC12-HSL-dipendente di Pseudomonas aeruginosa
Pseudobactin biogenesis in the plant growth-promoting rhizobacterium Pseudomonas strain B10: Identification and functional analysis of the L-Ornithine N-5-oxygenase (psbA) gene
No full textPseudobactin(B10), the fluorescent siderophore produced by the rhizobacterium Pseudomonas strain B10, contains the hydroxamate ligand D-N-5-hydroxyornithine (D-N-5-OH-Orn). We cloned the L-Orn N-5-oxygenase (psbA) gene from a genomic library of Pseudomonas strain B10 and demonstrated that PsbA is involved in the conversion of L-Orn to its N-5-OH derivative. PsbA shows significant similarity to microbial omega-amino acid hydroxylases containing flavin adenine dinucleotide and NADP cofactor-binding sites and the FATGY signature of the putative substrate recognition pocket. The psbA gene is monocistronic, and its transcription is negatively controlled by iron. A site-specific psbA mutant of Pseudomonas strain B10 was biochemically complemented with the precursor L-N-5-OH-Orn, suggesting that L-Orn is hydroxylated before conversion to the D isomer. The L-Orn N-5-hydroxylase-defective mutants of Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1 were much less effective than the parental strains in suppressing the growth of the phytopathogen Erwinia carotovora in iron-poor medium. The extent of in vitro inhibition of E. carotovora was strictly iron dependent and directly correlated with the amount of released siderophores. These data strengthen the role of fluorescent siderophores in biocontrol of deleterious rhizomicroorganisms
The interplay of StyR and IHF regulates substrate-dependent induction and carbon catabolite repression of styrene catabolism genes in Pseudomonas fluorescens ST
No full textBackground: In Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and is subject to catabolite repression. PstyA regulation relies on the StyS/ StyR two-component system and on the IHF global regulator. The phosphorylated response regulator StyR (StyR-P) activates PstyA in inducing conditions when it binds to the high-affinity site STY2, located about -40 bp from the transcription start point. A cis-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 PstyA catabolite repression could rely on the interplay of these regulators.
Results: StyR-P and IHF compete for binding to the URE region. PstyA 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 PstyA repression is achieved through an increase in the StyR-P/StyR ratio.
Conclusion: We propose a model according to which the activity of the PstyA 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 PstyA catabolite repression is likely the kinase activity of the StyR-cognate sensor protein StyS
Dual role of response regulator StyR in styrene catabolism regulation
No full textIn Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and repressed by the addition of preferred carbon sources. PstyA is regulated by the StyS/StyR two-component system. The integration host factor (IHF) also plays a positive role in PstyA regulation. Three distinct StyR binding sites, which have different affinities for this response regulator, have been characterized on PstyA. The high-affinity StyR binding site (STY2) is necessary for promoter activity. The DNA region upstream of STY2 contains a lower-affinity StyR binding site, STY1, that partially overlaps the IHF binding site. Deletion of this region, designated URE (upstream regulatory element), has a dual effect on the PstyA promoter, decreasing the styrene-dependent activity and partially relieving the glucose repression. The lowest-affinity StyR binding site (STY3) is located downstream of the transcription start point. Deletion of the URE region and inactivation of the STY3 site completely abolished glucose-mediated repression of PstyA. In the proposed model StyR can act either as an activator or as a repressor, depending on which sites it occupies in the different growth conditions. We suggest that the cellular levels of phosphorylated StyR, as determined by StyS sensor kinase activity, and the interplay of this molecule with IHF modulate the activity of the promoter in different growth conditions
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