Pyranoanthocyanins Interfering with the Quorum Sensing of Pseudomonas aeruginosa and Staphylococcus aureus.
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ABSTRACT:
SUBMITTER: Coelho P
PROVIDER: S-EPMC8395250 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Several serious diseases are caused by biofilm-associated Staphylococcus aureus, infections in which the accessory gene regulator (agr) quorum-sensing system is thought to play an important role. We studied the contribution of agr to biofilm development, and we examined agr-dependent transcription in biofilms. Under some conditions, disruption of agr expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where agr expression enhanced biofilm formation, biofilms of an agr signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an agr-independent fluorescent reporter, biofilm expression of an agr-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of agr expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing agr from biofilms may have important clinical implications.
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Project description:In a process termed quorum sensing, bacteria use diffusible chemical signals to coordinate cell density-dependent gene expression. In the human pathogen Pseudomonas aeruginosa, quorum sensing controls hundreds of genes, many of which encode extracellular virulence factors. Quorum sensing is required for P. aeruginosa virulence in animal models. Curiously, quorum sensing-deficient variants, most of which carry a mutation in the gene encoding the central quorum sensing regulator lasR, are frequently isolated from acute and chronic infections. The mechanism for their emergence is not known. Here we provide experimental evidence suggesting that these lasR mutants are social cheaters that cease production of quorum-controlled factors and take advantage of their production by the group. We detected an emerging subpopulation of lasR mutants after approximately 100 generations of in vitro evolution of the P. aeruginosa wild-type strain under culture conditions that require quorum sensing for growth. Under such conditions, quorum sensing appears to impose a metabolic burden on the proliferating bacterial cell, because quorum-controlled genes not normally induced until cessation of growth were highly expressed early in growth, and a defined lasR mutant showed a growth advantage when cocultured with the parent strain. The emergence of quorum-sensing-deficient variants in certain environments is therefore an indicator of high quorum sensing activity of the bacterial population as a whole. It does not necessarily indicate that quorum sensing is insignificant, as has previously been suggested. Thus, novel antivirulence strategies aimed at disrupting bacterial communication may be particularly effective in such clinical settings.
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Project description:The bacterial pathogen Pseudomonas aeruginosa activates expression of many virulence genes in a cell density-dependent manner by using an intricate quorum-sensing (QS) network. QS in P. aeruginosa involves two acyl-homoserine-lactone circuits, LasI-LasR and RhlI-RhlR. LasI-LasR is required to activate many genes including those coding for RhlI-RhlR. P. aeruginosa causes chronic infections in the lungs of people with cystic fibrosis (CF). In these infections, LasR mutants are common, but rhlR-rhlI expression has escaped LasR regulation in many CF isolates. To better understand the evolutionary trajectory of P. aeruginosa QS in chronic infections, we grew LasR mutants of the well-studied P. aeruginosa strain, PAO1, in conditions that recapitulate an environment where QS signal synthesis by other bacteria might still occur. When QS is required for growth, addition of the RhlI product butyryl-homoserine lactone (C4-HSL), or bacteria that produce C4-HSL, to LasR mutants results in the rapid emergence of a population with a LasR-independent RhlI-RhlR QS system. These evolved populations exhibit subsequent growth without added C4-HSL. The variants that emerge have mutations in mexT, which codes for a transcription factor that controls expression of multiple genes. LasR-MexT mutants have a competitive advantage over both the parent LasR mutant and a LasR-MexT-RhlR mutant. Our findings suggest a plausible evolutionary trajectory for QS in P. aeruginosa CF infections where LasR mutants arise during infection, but because these mutants are surrounded by C4-HSL-producing P. aeruginosa, variants rewired to have a LasR-independent RhlIR system quickly emerge.
| S-EPMC6452656 | biostudies-literature
Project description:In the pathogen Pseudomonas aeruginosa, LasR is a quorum sensing (QS) master regulator that senses the concentration of secreted autoinducers as a proxy for bacterial cell density. Counterintuitively, previous studies showed that saturating amounts of the LasR ligand, 3OC12-HSL, fail to induce the full LasR regulon in low-density liquid cultures. Here we demonstrate that surface association, which is necessary for many of the same group behaviors as QS, promotes stronger QS responses. We show that lasR is upregulated upon surface association, and that surface-associated bacteria induce LasR targets more strongly in response to autoinducer than planktonic cultures. This increased sensitivity may be due to surface-dependent lasR induction initiating a positive feedback loop through the small RNA, Lrs1. The increased sensitivity of surface-associated cells to QS is affected by the type IV pilus (TFP) retraction motors and the minor pilins. The coupling of physical surface responses and chemical QS responses could enable these bacteria to trigger community behaviors more robustly when they are more beneficial.
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Project description:Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels.IMPORTANCEPseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.
| S-EPMC6456851 | biostudies-literature
Project description:This manuscript describes the fabrication of arrays of spatially confined chambers embossed in a layer of poly(ethylene glycol) diacrylate (PEGDA) and their application to studying quorum sensing between communities of Pseudomonas aeruginosa. We hypothesized that biofilms may produce stable chemical signaling gradients in close proximity to surfaces, which influence the growth and development of nearby microcolonies into biofilms. To test this hypothesis, we embossed a layer of PEGDA with 1.5-mm wide chambers in which P. aeruginosa biofilms grew, secreted homoserine lactones (HSLs, small molecule regulators of quorum sensing), and formed spatial and temporal gradients of these compounds. In static growth conditions (i.e., no flow), nascent biofilms secreted N-(3-oxododecanoyl) HSL that formed a gradient in the hydrogel and was detected by P. aeruginosa cells that were ?8 mm away. Diffusing HSLs increased the growth rate of cells in communities that were <3 mm away from the biofilm, where the concentration of HSL was >1 ?M, and had little effect on communities farther away. The HSL gradient had no observable influence on biofilm structure. Surprisingly, 0.1-10 ?M of N-(3-oxododecanoyl) HSL had no effect on cell growth in liquid culture. The results suggest that the secretion of HSLs from a biofilm enhances the growth of neighboring cells in contact with surfaces into communities and may influence their composition, organization, and diversity.
| S-EPMC3076519 | biostudies-literature
Project description:During infection, Pseudomonas aeruginosa employs bacterial communication (quorum sensing [QS]) to coordinate the expression of tissue-damaging factors. QS-controlled gene expression plays a pivotal role in the virulence of P. aeruginosa, and QS-deficient mutants cause less severe infections in animal infection models. Treatment of cystic fibrosis (CF) patients chronically infected with P. aeruginosa with the macrolide antibiotic azithromycin (AZM) has been demonstrated to improve the clinical outcome. Several studies indicate that AZM may accomplish its beneficial action in CF patients by impeding QS, thereby reducing the pathogenicity of P. aeruginosa. This led us to investigate whether QS inhibition is a common feature of antibiotics. We present the results of a screening of 12 antibiotics for their QS-inhibitory activities using a previously described QS inhibitor selector 1 strain. Three of the antibiotics tested, AZM, ceftazidime (CFT), and ciprofloxacin (CPR), were very active in the assay and were further examined for their effects on QS-regulated virulence factor production in P. aeruginosa. The effects of the three antibiotics administered at subinhibitory concentrations were investigated by use of DNA microarrays. Consistent results from the virulence factor assays, reverse transcription-PCR, and the DNA microarrays support the finding that AZM, CFT, and CPR decrease the expression of a range of QS-regulated virulence factors. The data suggest that the underlying mechanism may be mediated by changes in membrane permeability, thereby influencing the flux of N-3-oxo-dodecanoyl-L-homoserine lactone.
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Project description:The opportunistic bacterial pathogen Pseudomonas aeruginosa has a layered acyl-homoserine lactone (AHL) quorum-sensing (QS) system, which controls production of a variety of extracellular metabolites and enzymes. The LasRI system activates genes including those coding for the extracellular protease elastase and for the second AHL QS system, RhlRI. Growth of P. aeruginosa on casein requires elastase production and LasR-mutant social cheats emerge in populations growing on casein. P. aeruginosa colonizes the lungs of individuals with the genetic disease cystic fibrosis (CF), and LasR mutants can be isolated from the colonized lungs; however, unlike laboratory-generated LasR mutants, many of these CF isolates have functioning RhlR-RhlI systems. We show that one such mutant can use the RhlR-RhlI system to activate expression of elastase and grow on casein. We carried out social-evolution experiments by growing this isolate on caseinate and, as with wild-type P. aeruginosa, elastase-negative mutants emerge as cheats, but these are not RhlR mutants; rather, they are mutants that do not produce the non-AHL Pseudomonas quinolone signal (PQS). Furthermore, we generated a RhlRI mutant and showed it had a fitness defect when growing together with the parent. Apparently, RhlR QS and PQS collude to support growth on caseinate in the absence of a functional LasR. Our findings provide a plausible explanation as to why P. aeruginosa LasR mutants, but not RhlR mutants, are common in CF lungs.
| S-EPMC6452681 | biostudies-literature
Project description:The opportunistic pathogen Pseudomonas aeruginosa utilizes two interconnected acyl-homoserine lactone quorum-sensing (acyl-HSL QS) systems, LasRI and RhlRI, to regulate the expression of hundreds of genes. The QS circuitry itself is integrated into a complex network of regulation by other factors. However, our understanding of this network is still unlikely to be complete, as a comprehensive, saturating approach to identifying regulatory components has never been attempted. Here, we utilized a nonredundant P. aeruginosa PA14 transposon library to identify additional genes that regulate QS at the level of LasRI/RhlRI. We initially screened all 5,459 mutants for loss of function in one QS-controlled trait (skim milk proteolysis) and then rescreened attenuated candidates for defects in other QS phenotypes (LasA protease, rhamnolipid, and pyocyanin production) to exclude mutants defective in functions other than QS. We identified several known and novel genes, but only two novel genes, gidA and pcnB, affected all of the traits assayed. We characterized gidA, which exhibited the most striking QS phenotypes, further. This gene is predicted to encode a conserved flavin adenine dinucleotide-binding protein involved in tRNA modification. Inactivation of the gene primarily affected rhlR-dependent QS phenotypes such as LasA, pyocyanin, and rhamnolipid production. GidA affected RhlR protein but not transcript levels and also had no impact on LasR and acyl-HSL production. Overexpression of rhlR in a gidA mutant partially restored QS-dependent phenotypes. Taken together, these results indicate that GidA selectively controls QS gene expression posttranscriptionally via RhlR-dependent and -independent pathways.
| S-EPMC2737955 | biostudies-literature
Project description:Quorum sensing (QS) is a chemical communication process that Pseudomonas aeruginosa uses to regulate virulence and biofilm formation. Disabling of QS is an emerging approach for combating its pathogenicity. Silver nanoparticles (AgNPs) have been widely applied as antimicrobial agents against human pathogenic bacteria and fungi, but not for the attenuation of bacterial QS. Here we mycofabricated AgNPs (mfAgNPs) using metabolites of soil fungus Rhizopus arrhizus BRS-07 and tested their effect on QS-regulated virulence and biofilm formation of P. aeruginosa. Transcriptional studies demonstrated that mfAgNPs reduced the levels of LasIR-RhlIR. Treatment of mfAgNPs inhibited biofilm formation, production of several virulence factors (e.g. LasA protease, LasB elastrase, pyocyanin, pyoverdin, pyochelin, rhamnolipid, and alginate) and reduced AHLs production. Further genes quantification analyses revealed that mfAgNPs significantly down-regulated QS-regulated genes, specifically those encoded to the secretion of virulence factors. The results clearly indicated the anti-virulence property of mfAgNPs by inhibiting P. aeruginosa QS signaling.
| S-EPMC4562228 | biostudies-literature