Browsing by Subject "quorum sensing"

Sort by: Order: Results:

Now showing items 1-10 of 10
  • Gatta, Viviana; Tomašič, Tihomir; Ilaš, Janez; Zidar, Nace; Peterlin Mašič, Lucija; Barančoková, Michaela; Frlan, Rok; Anderluh, Marko; Kikelj, Danijel; Tammela, Päivi (2020)
    Quorum sensing (QS), a bacterial communication strategy, has been recognized as one of the control mechanisms of virulence in bacteria. Thus, targeting QS offers an interesting opportunity to impair bacterial pathogenicity and develop antivirulence agents. Aiming to enhance the discovery of QS inhibitors, we developed a bioreporter Escherichia coli JW5505 pET-Plsrlux and set up a cell-based assay for identifying inhibitors of autoinducer-2 (AI-2)-mediated QS. A comparative study on the performance of target- versus cell-based assays was performed, and 91 compounds selected with the potential to target the ATP binding pocket of LsrK, a key enzyme in AI-2 processing, were tested in an LsrK inhibition assay, providing 36 hits. The same set of compounds was tested by the AI-2-mediated QS interference assay, resulting in 24 active compounds. Among those, six were also found to be active against LsrK, whereas 18 might target other components of the pathway. Thus, this AI-2-mediated QS interference cell-based assay is an effective tool for complementing target-based assays, yet also stands as an independent assay for primary screening.
  • Stotani, Silvia; Gatta, Viviana; Medda, Federico; Padmanaban, Mohan; Karawajzyk, Anna; Tammela, Päivi Sirpa Marjaana; Giordanetto, Fabrizio; Tzalis, Dimitrios; Collina, Simona (2018)
    Resistance to antibiotics is an increasingly serious threat to global public health and its management translates to significant health care costs. The validation of new Gram-negative antibacterial targets as sources for potential new antibiotics remains a challenge for all the scientists working in this field. The interference with bacterial Quorum Sensing (QS) mechanisms represents a potentially interesting approach to control bacterial growth and pursue the next generation of antimicrobials. In this context, our research is focused on the discovery of novel compounds structurally related to (S)-4,5-dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small signaling molecule able to modulate bacterial QS in both Gram-negative and Gram-positive bacteria. In this study, a practical and versatile synthesis of racemic DPD is presented. Compared to previously reported syntheses, the proposed strategy is short and robust: it requires only one purification step and avoids the use of expensive or hazardous starting materials as well as the use of specific equipment. It is therefore well suited to the synthesis of derivatives for pharmaceutical research, as demonstrated by four series of novel DPD-related compounds described herein.
  • Kaya, Meryem Ecem (Helsingin yliopisto, 2019)
    Synthetic biology is an emerging interdisciplinary field of biology that aims to system-atically design artificial biological systems. As synthetic biologists seek increasingly complex control over cellular processes to achieve robust and predictable systems. A new frontier in synthetic biology is engineering synthetic microbial consortia. This ap-proach employs the concept of division of labor, instead of introducing large genetic cir-cuitry to homogenous cell populations. In this approach, different cell types are assigned to execute a portion of the overall circuit. Each cell type communicates with their co-worker subpopulations to complete the circuit. The main advantage of this strategy is the reduced metabolic burden on each cell type. Thus, leading to more reliable and stable overall performance. In this work, to simplify cellular communication between the mem-bers of the consortium, we used the simple architecture of quorum sensing machinery. We constructed a toolbox that contains promoter, receptor and quorum sensing signal synthase genes along with fluorescent reporters. Using this toolbox, we constructed dif-ferent cell types that can be used in synthetic consortia forming various communication topologies. We characterized the constructed cell types individually and in co-cultures.
  • Skogman, Malena E.; Kanerva, Sonja; Manner, Suvi; Vuorela, Pia M.; Fallarero, Adyary (2016)
    Quorum sensing (QS) is the process by which bacteria produce and detect signal molecules to coordinate their collective behavior. This intercellular communication is a relevant target for anti-biofilm therapies. Here we have optimized a screening-applicable assay to search for new quorum sensing inhibitors from natural compound libraries. In this system, QS is correlated with the production of violacein, which is directly controlled by the LuxI/LuxR system in Chromobacterium violaceum ATCC 31532. The parallel use of C. violaceum Tn5-mutant CV026, which depends on auto-inducer addition, allows simultaneous discrimination of compounds that act as quenchers of the AHL signal (quorum quenchers). The incorporation of a redox stain into the platform allowed further distinction between QS inhibitors, quorum quenchers and antibacterial compounds. A pilot screening was performed with 465 natural and synthetic flavonoids. All the most active compounds were flavones and they displayed potencies (IC50) in the range of 3.69 to 23.35 M. These leads were particularly promising as they inhibited the transition from microcolonies into mature biofilms from Escherichia coli and Pseudomonas aeruginosa strains. This approach can be very effective in identifying new antimicrobials posing lesser risks of resistance.
  • Mäkkylä, Heidi (Helsingfors universitet, 2017)
    Bacteria can communicate with each other using phenomenon called quorum sensing (QS). In QS the bacteria produce and release small signaling molecules which they use to communicate. Bacteria use QS in situations where it is beneficial to act on population level. QS has an important role e.g. in the formation of virulence factors and biofilms. There are several different QS systems. Gram-negative bacteria use i.a AI-1, AI-2, AI-3, and CAI-1 systems to communicate. All QS systems are based on the accumulation of signaling molecules when the bacterial concentration increases. When the concentration of signal molecules reaches the threshold level, the system activates. The activation of the signaling system then activates the expression of the genes controlled by the QS system. AI-2 signaling is assumed to be universal. That means that bacteria can use AI-2 signaling system in interspecies communication. In AI-2 signaling bacteria produce and release 4,5-dihydroxy-2,3-pentanedione (DPD) which works as a signaling molecule in the AI-2 system. Escherichia coli and Salmonella typhimurium use an ATP binding cassette ABC-type transporter to transport DPD molecules into the cell where LsrK kinase phosphorylates the DPD molecules. The phosphorylated DPD molecules bind to the LsrR regulator protein which acts as a suppressor of the lsr operon. The binding of the phosphorylated DPD molecules releases the LsrR from the lsr promoter region enabling the expression of the lsr genes. In Vibrio harveyi the surface proteins LuxP and LuxQ form a protein complex that recognizes DPD molecules. When the DPD concentration increases, the LuxPQ complex transform from kinase to phosphatase and the reaction chain, where LuxU phosphate transfer protein transfers a phosphate group from LuxO regulator protein, activates. The dephosphorylation of of LuxO releases the LuxR transcription factor and activates the expression of QS controlled genes. The aim of this thesis was to optimize two assays which can be used to screen for compounds that disrupt AI-2 signaling. The first assay was a bioreporter based assay where V. harveyi BB120 bioreporter strain was used. The second assay was protein based LsrK assay where the LsrK activity was monitored using assay kit which measures the concentration of ATP or ADP. The concentrations of bacteria, LsrK, and DPD used in the assays were optimized. The dimethyl sulfoxide (DMSO) tolerance of both assays were tested, the stability of the kits used in the LsrK assays was tested and the reaction buffer for the LsrK assay was selected from the two tested buffer options. The selected bacterial concentration for the V. harveyi BB120 assay was 100000 CFU/ml and DPD concentration 1 µM. The selected enzyme concentration for the LsrK assay was 300 nM and DPD concentration 300 µM. The tested DMSO concentrations had no effect on the kit measuring ATP but the highest concentrations tested had a small effect on the kit measuring ADP. A buffer containing triethanolamine, magnesium chloride, and bovine serum albumin was selected as the reaction buffer for the LsrK assay. Using the optimized LsrK assay, a screening was performed for a synthesized compound library. None of the compounds showed any LsrK inhibiting activity. The optimized assay was also used to make dose-response experiment to one LsrK inhibiting compound, named FIMM000642, which was found in a separate screening. The FIMM000642 dose-response as-say was also done against glycerol kinase to see if the compound would inhibit another enzyme from the same protein family or if the compound was a specific inhibitor to LsrK. FIMM000642 inhibited also the activity of glycerol kinase.
  • Pitkänen, Katriina (Helsingin yliopisto, 2022)
    Bacteria coordinate the virulence factor production and biofilm formation by activating the mechanism of intercellular communication called quorum sensing (QS). The biofilm matrix protects bacteria against variety of stress conditions, including e.g., effects of antibiotics and disinfectants. QS inhibitors (QSI) are molecules that can inhibit the QS mechanism. QQ is a QSI inhibitor that interrupts the cell-to-cell signaling already outside of the bacterial cell. Dairy propionic acid bacteria have known antimicrobial properties but their ability to produce anti-QS compounds have not yet been studied. Previous studies have shown positive results of the anti-QS activity exerted by lactobacilli, but more research is needed to uncover mechanistic details behind their QS-inhibitory effects. The aim of this study was to investigate the anti-QS and QQ activity of propionic bacteria and lactobacilli. The used method was an in-house optimized screening method based on using wild-type and mutant strains of Chromobacterium violaceum. This bioreporter produces a QS-mediated, measurable color pigment. Here, the limit of potential QSI and QQ inhibition was set to 50 % inhibition. Viability of bioreporter was analyzed by resazurin staining. Propionibacterium freudenreichii strains demonstrated both the QSI (64–71%) and QQ (52–60%) activities against the tested C. violaceum reporters. In the case of lactobacilli, Lacticaseibacillus rhamnosus GG, Limosilactobacillus mucosae 2674, Lactobacillus acidophilus 80, Lactobacillus curvatus 453 and Lactobacillus kefiri 3070 inhibited the QS-activation of the wild-type bioreporter strain by 45 to 90 %. There was a great variability in the anti-QS screening assays involving the mutant strain, which may be explained by the possible sensitivity of the compounds to the analysis conditions used or more sensitive response of the mutant strain to the tested agents. In conclusion, the obtained results show that L. acidophilus 80 produced bactericidal compounds. L. reuteri 410 did not have any effect on the QS-indicator production in wild-type bioreporter but was able to inhibit QS-induction in the mutant reporter by 72% (QQ activity). This study showed that P. freudenreichii strains as well as certain lactobacilli have an ability to produce QSI and QQ activity against some gram-negative biofilm pathogens. Pathogens have different QS mechanisms and experiments with C. violaceum strains are only one way to screen for anti-QS compounds. Therefore, validation of the obtained findings requires complementary screening assays against other bacterial bioreporters, involving both gram-negative and gram-positive biofilm bacteria.
  • Gilbert-Girard, Shella; Savijoki, Kirsi; Yli-Kauhaluoma, Jari; Fallarero, Adyary (2020)
    In an effort to find new repurposed antibacterial compounds, we performed the screening of an FDA-approved compounds library against Staphylococcus aureus American Type Culture Collection (ATCC) 25923. Compounds were evaluated for their capacity to prevent both planktonic growth and biofilm formation as well as to disrupt pre-formed biofilms. One of the identified initial hits was fingolimod (FTY720), an immunomodulator approved for the treatment of multiple sclerosis, which was then selected for follow-up studies. Fingolimod displayed a potent activity against S. aureus and S. epidermidis with a minimum inhibitory concentration (MIC) within the range of 12-15 mu M at which concentration killing of all the bacteria was confirmed. A time-kill kinetic study revealed that fingolimod started to drastically reduce the viable bacterial count within two hours and we showed that no resistance developed against this compound for up to 20 days. Fingolimod also displayed a high activity against Acinetobacter baumannii (MIC 25 mu M) as well as a modest activity against Escherichia coli and Pseudomonas aeruginosa. In addition, fingolimod inhibited quorum sensing in Chromobacterium violaceum and might therefore target this signaling pathway in certain Gram-negative bacteria. In conclusion, we present the identification of fingolimod from a compound library and its evaluation as a potential repurposed antibacterial compound.
  • Manner, Suvi; Fallarero, Adyary (2018)
    Owing to the failure of conventional antibiotics in biofilm control, alternative approaches are urgently needed. Inhibition of quorum sensing (QS) represents an attractive target since it is involved in several processes essential for biofilm formation. In this study, a compound library of natural product derivatives (n = 3040) was screened for anti-quorum sensing activity using Chromobacterium violaceum as reporter bacteria. Screening assays, based on QS-mediated violacein production and viability, were performed in parallel to identify non-bactericidal QS inhibitors (QSIs). Nine highly active QSIs were identified, while 328 compounds were classified as moderately actives and 2062 compounds as inactives. Re-testing of the highly actives at a lower concentration against C. violaceum, complemented by a literature search, led to the identification of two flavonoid derivatives as the most potent QSIs, and their impact on biofilm maturation in Escherichia coli and Pseudomonas aeruginosa was further investigated. Finally, effects of these leads on swimming and swarming motility of P. aeruginosa were quantified. The identified flavonoids affected all the studied QS-related functions at micromolar concentrations. These compounds can serve as starting points for further optimization and development of more potent QSIs as adjunctive agents used with antibiotics in the treatment of biofilms.
  • Yang, Weifeng; Wei, Qing; Tong, Qian; Cui, Kaiyu; He, Gaiying; Lin, Longfei; Ma, Lvyan Z.; Cornelis, Pierre; Wang, Yi (2020)
    Pseudomonas aeruginosa is an opportunistic pathogen that can infect a wide variety of hosts including humans, plants, and animals. The production of virulence factors is the determinant of the infection paradigm and is under orchestrated regulation via cell-to-cell communication process called quorum sensing (QS). To disable QS circuits and prevent bacterial infections, a large battery of anti-QS agents, particularly from traditional Chinese medicine have been developed. Here, we used P. aeruginosa as a model microorganism to investigate the effect of traditional Chinese medicine Tanreqing (TRQ) formula on bacterial pathogenicity. Phenotypic analysis showed that TRQ treatment could completely inhibit the production of phenazine pyocyanin and moderately inhibit the production of virulence factors such as rhamnolipids, elastase, and alkaline protease. Further transcriptomic analyses revealed that TRQ treatment could significantly attenuate the expression of QS-regulated genes in P. aeruginosa and TRQ-treated P. aeruginosa regulon shared a large overlap with QS regulon. Component contribution to QS inhibition shed light on the indispensable role of all five components in TRQ formula. Further genetic analysis indicated that upstream regulators of QS systems, including two-component systems GacS/GacA and PprA/PprB, were both inhibited by TRQ treatment. Finally, our TRQ formula could efficiently protect Caenorhabditis elegans from killing by P. aeruginosa. Altogether, we have proved TRQ formula as an effective and specific agent to attenuate bacterial virulence and combat bacterial infections.
  • Nicol, Marion; Alexandre, Stephane; Luizet, Jean-Baptiste; Skogman, Malena; Jouenne, Thierry; Salcedo, Suzana P.; De, Emmanuelle (2018)
    The increasing threat of Acinetobacter baumannii as a nosocomial pathogen is mainly due to the occurrence of multidrug-resistant strains that are associated with the real problem of its eradication from hospital wards. The particular ability of this pathogen to form biofilms contributes to its persistence, increases antibiotic resistance, and promotes persistent/device-related infections. We previously demonstrated that virstatin, which is a small organic compound known to decrease virulence of Vibrio cholera via an inhibition of T4-pili expression, displayed very promising activity to prevent A. baumannii biofilm development. Here, we examined the antibiofilm activity of mono-unsaturated chain fatty acids, palmitoleic (PoA), and myristoleic (MoA) acids, presenting similar action on V. cholerae virulence. We demonstrated that PoA and MoA (at 0.02 mg/mL) were able to decrease A. baumannii ATCC 17978 biofilm formation up to 38% and 24%, respectively, presented a biofilm dispersing effect and drastically reduced motility. We highlighted that these fatty acids decreased the expression of the regulator abaR from the LuxIR-type quorum sensing (QS) communication system AbaIR and consequently reduced the N-acyl-homoserine lactone production (AHL). This effect can be countered by addition of exogenous AHLs. Besides, fatty acids may have additional non-targeted effects, independent from QS. Atomic force microscopy experiments probed indeed that PoA and MoA could also act on the initial adhesion process in modifying the material interface properties. Evaluation of fatty acids effect on 22 clinical isolates showed a strain-dependent antibiofilm activity, which was not correlated to hydrophobicity or pellicle formation ability of the tested strains, and suggested a real diversity in cell-to-cell communication systems involved in A. baumannii biofilm formation.