Quorum-sensing Receptor Research Articles

Unveiling Quorum Sensing Mechanisms: Computational Docking and Dynamics of Bacterial Receptors and Ligands

The bacterial cell-to-cell communication mechanism known as quorum sensing (QS) uses chemical cues called autoinducers (AIs) to control several processes, including pathogenicity, antibiotic resistance, and biofilm formation. This study investigates the QS receptor-ligand interactions and QS compatibility within and between bacterial species using computational molecular docking. Receptor proteins including LuxS, LuxP, AgrC, LuxN, SdiA, LasR, esaR, YenR, LamC, PlcR, and TraR were docked with their respective AIs (AHL, AI-1, AI-2, AIP1, LamD, PapR7) in both biofilm and non-biofilm producing bacteria. Our findings indicate that QS receptors exhibit high affinity for their cognate ligands, with binding affinities ≥ -4.5 Kcal/mol. Additionally, Zinc Pharmar-derived similar chemical structures demonstrated binding affinities ≥ -5.3 Kcal/mol. Density Functional Theory (DFT) analysis revealed AI-2 as the most reactive autoinducer. Molecular Dynamics (MD) simulations confirmed the stability of LasR-AHL and LuxP-AI-2 complexes. These insights pave the way for further in vitro and in vivo investigations of QS mechanisms.

Synthesis, Antibacterial, Anti-Biofilm Properties, and Docking Study of Indeno[1,2-b]Pyridin-5-One Derivatives

In this context, we designed and synthesized a series of hydrazonal and their related indeno[1,2-b]pyridin-5-one derivatives to investigate their antibacterial and anti-biofilm properties. Several of the synthesized compounds exhibited significant efficacy against all microorganism species tested. Most of the compounds demonstrated favorable results when tested against Gram-positive bacteria. The derivatives 4a, 4f, 6c, and 6f exhibit the highest antimicrobial efficacy, as indicated by their minimum inhibitory concentration (MIC) values ranging from 4 to 512 μg/mL. We conducted additional investigations on 4a, 6c, and 6f for the purpose of examining their synergy using Checkerboard assay. Compound 6c demonstrated a synergistic impact against methicillin-sensitive Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa, while exhibiting moderate synergistic activity against methicillin-resistant Staphylococcus aureus (MRSA). In addition, the simultaneous use of gentamycin with compounds 4a and 6f demonstrates a synergistic impact in fighting against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, respectively. Three chosen compounds were evaluated for their antibiofilm efficacy. Application of 4a, 6c, and 6f effectively reduced the production of biofilms in MRSA, MSSA, and Pseudomonas aeruginosa, resulting in a significant decrease compared to the untreated samples. In addition, ADME and pharmacokinetic analyses were conducted for the three most potent derivatives, namely 4a, 6c, and 6f. Compounds 4a and 6c were subjected to docking in the LasR Quorum-Sensing Receptor, whereas compounds 6c and 6f were docked in the sortase enzyme.

Endophytic Penicillium oxalicum AUMC 14898 from Opuntia ficus-indica: A Novel Source of Tannic Acid Inhibiting Virulence and Quorum Sensing of Extensively Drug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a harmful pathogen that causes a variety of acute and chronic infections through quorum sensing (QS) mechanisms. The increasing resistance of this bacterium to numerous antibiotics has created a demand for new medications that specifically target QS. Endophytes can be the source of compounds with antibacterial properties. This research is the first to examine tannic acid (TA) produced by endophytic fungus as a potential biotherapeutic agent. A novel endophytic fungal isolate identified as Penicillium oxalicum was derived from the cladodes of Opuntia ficus-indica (L.). The species identification for this isolate was confirmed through sequencing of the internal transcribed spacer region. The metabolites from the culture of this isolate were extracted using ethyl acetate, then separated and characterized using chromatographic methods. This led to the acquisition of TA, a compound that shows strong anti-QS and excellent antibacterial effects against extensively drug-resistant P. aeruginosa strains. Furthermore, it was shown that treating P. aeruginosa with the obtained TA reduced the secretion of virulence factors controlled by QS in a dose-dependent manner, indicating that TA inhibited the QS characteristics of P. aeruginosa. Simultaneously, TA significantly inhibited the expression of genes associated with QS, including rhlR/I, lasR/I, and pqsR. In addition, in silico virtual molecular docking showed that TA could efficiently bind to QS receptor proteins. Our results showed that P. oxalicum could be a new source of TA for the treatment of infections caused by extensively drug-resistant P. aeruginosa.

Transcriptomics analysis of the role of SdiA in desiccation tolerance of Cronobacter sakazakii in powdered infant formula

The quorum-sensing receptor SdiA is vital for regulating the desiccation tolerance of C. sakazakii, yet the specific mechanism remains elusive. Herein, transcriptomics and phenotypic analysis were employed to explore the response of C. sakazakii wild type (WT) and sdiA knockout strain (ΔsdiA) under drying conditions. Following 20 days of drying in powdered infant formula (PIF), WT exhibited 4 log CFU/g higher survival rates compared to ΔsdiA. Transcriptome revealed similar expression patterns between csrA and sdiA, their interaction was confirmed both by protein-protein interaction analysis and yeast two-hybrid assays. Notably, genes associated with flagellar assembly and chemotaxis (flg, fli, che, mot regulon) showed significantly higher expression levels in WT than in ΔsdiA, indicating a reduced capacity for flagellar synthesis in ΔsdiA, which was consistent with cellular morphology observations. Similarly, genes involved in trehalose biosynthesis (ostAB, treYZS) and uptake (thuEFGK) exhibited similar expression patterns to sdiA, with higher levels of trehalose accumulation observed in WT under desiccation conditions compared to ΔsdiA. Furthermore, WT demonstrated enhanced protein and DNA synthesis capabilities under desiccation stress. Higher expression levels of genes related to oxidative phosphorylation were also noted in WT, ensuring efficient cellular ATP synthesis. This study offers valuable insights into how SdiA influences the desiccation tolerance of C. sakazakii, paving the way for targeted strategies to inhibit and control this bacterium.

Cilostazol is a promising anti-pseudomonal virulence drug by disruption of quorum sensing

Resistance to antibiotics is a critical growing public health problem that desires urgent action to combat. To avoid the stress on bacterial growth that evokes the resistance development, anti-virulence agents can be an attractive strategy as they do not target bacterial growth. Quorum sensing (QS) systems play main roles in controlling the production of diverse virulence factors and biofilm formation in bacteria. Thus, interfering with QS systems could result in mitigation of the bacterial virulence. Cilostazol is an antiplatelet and a vasodilator FDA approved drug. This study aimed to evaluate the anti-virulence activities of cilostazol in the light of its possible interference with QS systems in Pseudomonas aeruginosa. Additionally, the study examines cilostazol’s impact on the bacterium’s ability to induce infection in vivo, using sub-inhibitory concentrations to minimize the risk of resistance development. In this context, the biofilm formation, the production of virulence factors and influence on the in vivo ability to induce infection were assessed in the presence of cilostazol at sub-inhibitory concentration. Furthermore, the outcome of combination with antibiotics was evaluated. Cilostazol interfered with biofilm formation in P. aeruginosa. Moreover, swarming motility, biofilm formation and production of virulence factors were significantly diminished. Histopathological investigation revealed that liver, spleen and kidney tissues damage was abolished in mice injected with cilostazol-treated bacteria. Cilostazol exhibited a synergistic outcome when used in combination with antibiotics. At the molecular level, cilostazol downregulated the QS genes and showed considerable affinity to QS receptors. In conclusion, Cilostazol could be used as adjunct therapy with antibiotics for treating Pseudomonal infections. This research highlights cilostazol’s potential to combat bacterial infections by targeting virulence mechanisms, reducing the risk of antibiotic resistance, and enhancing treatment efficacy against P. aeruginosa. These findings open avenues for repurposing existing drugs, offering new, safer, and more effective infection control strategies.

Harmine acts as a quorum sensing inhibitor decreasing the virulence and antibiotic resistance of Pseudomonas aeruginosa

BackgroundAs antimicrobial resistance (AMR) has become a global health crisis, new strategies against AMR infection are urgently needed. Quorum sensing (QS), responsible for bacterial communication and pathogenicity, is among the targets for anti-virulence drugs that thrive as one of the promising treatments against AMR infection.MethodsWe identified a natural compound, Harmine, through virtual screening based on three QS receptors of Pseudomonas aeruginosa (P. aeruginosa) and explored the effect of Harmine on QS-controlled and pathogenicity-related phenotypes including pyocyanin production, exocellular protease excretion, biofilm formation, and twitching motility of P. aeruginosa PA14. The protective effect of Harmine on Caenorhabditis elegans (C. elegans) and mice infection models was determined and the synergistic effect of Harmine combined with common antibiotics was explored. The underlaying mechanism of Harmine’s QS inhibitory effect was illustrated by molecular docking analysis, transcriptomic analysis, and target verification assay.ResultsIn vitro results suggested that Harmine possessed QS inhibitory effects on pyocyanin production, exocellular protease excretion, biofilm formation, and twitching motility of P. aeruginosa PA14, and in vivo results displayed Harmine’s protective effect on C. elegans and mice infection models. Intriguingly, Harmine increased susceptibility of both PA14 and clinical isolates of P. aeruginosa to polymyxin B and kanamycin when used in combination. Moreover, Harmine down-regulated a series of QS controlled genes associated with pathogenicity and the underlying mechanism may have involved competitively antagonizing autoinducers’ receptors LasR, RhlR, and PqsR.ConclusionsThis study shed light on the anti-virulence potential of Harmine against QS targets, suggesting the possible use of Harmine and its derivates as anti-virulence compounds.

Characterization of natural product inhibitors of quorum sensing reveals competitive inhibition of Pseudomonas aeruginosa RhlR by ortho-vanillin.

Quorum sensing (QS) regulates many aspects of bacterial pathogenesis and has attracted much interest as a target for anti-virulence therapies over the past 30 years, for example, antagonists of the LasR and RhlR QS receptors in Pseudomonas aeruginosa. Potent and selective QS inhibitors remain relatively scarce. However, natural products have provided a bounty of chemical scaffolds with anti-QS activities, but their molecular mechanisms are poorly characterized. The current study serves to fill this void by examining the activity of an important and wide-spread class of natural product QS modulators, benzaldehydes, and related derivatives, in LasR and RhlR. We demonstrate that ortho-vanillin can act as a competitive inhibitor of RhlR, a receptor that has emerged and may supplant LasR in certain settings as a target for P. aeruginosa QS control. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities.

Citral and triclosan synergistically silence quorum sensing and potentiate antivirulence response in Pseudomonas aeruginosa.

Among the ESKAPE pathogens, Pseudomonas aeruginosa is an extensively notorious superbug that causes difficult-to-treat infections. Since quorum sensing (QS) directly promotes pseudomonal virulence, targeting QS circuits is a promising approach for disarming phenotypic virulence. Hence, this study scrutinizes the anti-QS, antivirulence, and anti-biofilm potential of citral (CiT; phytochemical) and triclosan (TcN; disinfectant), alone and in combination, against P. aeruginosa PAO1/PA14. The findings confirmed synergism between CiT and TcN and revealed their quorum quenching (QQ) potential. At sub-inhibitory levels, CiT-TcN combination significantly impeded pyocyanin, total bacterial protease, hemolysin, and pyochelin production alongside inhibiting biofilm formation in P. aeruginosa. Moreover, the QQ and antivirulence potential of CiT and TcN was positively correlated by molecular docking studies that predicted strong associations of the drugs with QS receptors of P. aeruginosa. Collectively, the study identifies CiT-TcN as an effective drug combination that harbors QQ, antivirulence, and anti-biofilm prospects against P. aeruginosa.

Myrtus communis leaf compounds as novel inhibitors of quorum sensing-regulated virulence factors and biofilm formation: In vitro and in silico investigations

Antibiotic resistance of the Gram-negative bacterium Pseudomonas aeruginosa and its ability to form biofilm through the Quorum Sensing (QS) mechanism are important challenges in the control of infections caused by this pathogen. The extract of Myrtus communis (myrtle) showed strong anti-QS effect on Chromobacterium. violaceum 6267 by inhibiting 80 % of the production of violacein pigment at a sub-MIC concentration of 1/8 (31.25 μg/mL). In addition, the extract exhibited an inhibitory effect on virulence factors of P. aeruginosa PAO1 at half MIC (125 μg/mL), significantly reducing the formation of biofilms (72.02 %), the swarming activity (75 %), and the production of protease (61.83 %) and pyocyanin (97 %). The active fraction also downregulated the expression of selected regulatory genes involved in the biofilm formation and QS in the P. aeruginosa PAO1 strain. These genes included the autoinducer synthase genes (lasI and rhlI), the genes involved in the expression of their corresponding receptors (lasR and rhlR), and the pqsA genes. The analysis of the active fraction by HPLC/UV/MS and NMR allowed the identification of three phenolic compounds, 3,5-di-O-galloylquinic acid, myricetin 3-O-α-l-rhamnopyranoside (myricitrin), and myricetin 3-O-(2″-O-galloyl)-ß-d-galactopyranoside. In silico studies showed that 3,5-di-O-galloylquinic acid, with an affinity score of −9.20 kcal/mol, had the highest affinity to the active site of the CviR protein (3QP8), a QS receptor from C. violaceum. Additionally, myricetin 3-O-α-l-rhamnopyranoside (myricitrin) and myricetin 3-O-(2″-O-galloyl)-ß-d-galactopyranoside interact to a lesser extent with 3QP8. In conclusion, this study contributed significantly to the discovery of new QS inhibitors from M. communis leaves against resistant Gram-negative pathogens.

Assessing the antibacterial potential of 6-gingerol: Combined experimental and computational approaches

The rise of antibiotic resistance in bacteria is becoming a global concern, particularly due to the dwindling supply of new antibiotics. This situation mandates the discovery of new antimicrobial candidates. Plant-derived natural compounds have historically played a crucial role in the development of antibiotics, serving as a rich source of substances possessing antimicrobial properties. Numerous studies have supported the reputation of 6-gingerol, a prominent compound found in the ginger family, for its antibacterial properties. In this study, the antibacterial activities of 6-gingerol were evaluated against Gram-negative bacteria, Acinetobacter baumannii and Klebsiella pneumoniae, with a particular focus on the clinically significant Gram-negative Pseudomonas aeruginosa and Gram-positive bacteria Staphylococcus aureus. Furthermore, the anti-virulence activities were assessed in vitro, in vivo, and in silico. The current findings showed that 6-gingerol’s antibacterial activity is due to its significant effect on the disruption of the bacterial cell membrane and efflux pumps, as it significantly decreased the efflux and disrupted the cell membrane of S. aureus and P. aeruginosa. Furthermore, 6-gingerol significantly decreased the biofilm formation and production of virulence factors in S. aureus and P. aeruginosa in concentrations below MICs. The anti-virulence properties of 6-gingerol could be attributed to its capacity to disrupt bacterial virulence-regulating systems; quorum sensing (QS). 6-Gingerol was found to interact with QS receptors and downregulate the genes responsible for QS. In addition, molecular docking, and molecular dynamics (MD) simulation results indicated that 6-gingerol showed a comparable binding affinity to the co-crystalized ligands of different P. aeruginosa QS targets as well as stable interactions during 100 ns MD simulations. These findings suggest that 6-gingerol holds promise as an anti-virulence agent that can be combined with antibiotics for the treatment of severe infections.

Utilization of zein nano-based system for promoting antibiofilm and anti-virulence activities of curcumin against Pseudomonas aeruginosa

Abstract Bacterial biofilms contribute to increased pathogenesis and bacterial resistance. Biofilms can enhance pathogenicity by shielding bacteria from the immune system and antibiotics, and they are associated with persistent infections. Additionally, the antibiotic resistance mechanisms within biofilms make them challenging to treat, emphasizing the need for strategies to be addressed. Mitigating bacterial virulence is a promising strategy that could ease their eradication by host immunity without stressing bacteria to induce resistance. The merits of this strategy are augmented when using safe anti-virulence candidates in proper formulations. The current study aimed to evaluate the antibiofilm and anti-virulence efficacy of curcumin–zein nanoparticles against Pseudomonas aeruginosa. In vitro investigations were performed to assess the effect of nanoparticles on biofilm formation, bacterial motility, and production of virulence factors, including proteases, hemolysins, and pyocyanin, in comparison to bulk curcumin. Furthermore, the effect on the expression of the genes that encode quorum sensing (QS) systems that regulate bacterial virulence was assessed. An in silico study was done to evaluate the affinity of curcumin to QS receptors. Additionally, an in vivo protection assay was performed to evaluate the inhibitory effect of our preparation on diminishing the P. aeruginosa’s capacity to induce pathogenesis. The results showed significant antibiofilm and anti-virulence activities of the curcumin–zein nanoparticles compared to bulk curcumin. These anti-virulence activities were attributed to the curcumin’s interfering with the P. aeruginosa QS systems that regulate its virulence. In conclusion, curcumin acquires significant anti-QS, anti-virulence, and antibiofilm activities that are vastly enhanced upon loading on zein nanoparticles.

Design, synthesis and exploration of novel triazinoindoles as potent quorum-sensing inhibitors and radical quenchers.

Background: Antimicrobial resistance has become a critical health concern, and quorum-sensing exacerbates the resistance by facilitating cell-to-cell communication within the microbial community, leading to severe pathogenic outbreaks. Methods & results: Novel 1-(2-((5H-[1,2,4]-triazino[5,6-b]indol-3-yl)thio)acetyl)indoline-2,3-diones were synthesized. The title compounds exhibit outstanding anti-quorum-sensing efficacy, and compound 7g demonstrated the maximum proficiency (IC50=0.0504μg/ml). The hybrids displayed potent antioxidant action, and compound 7c showed the highest antioxidant ability (IC50=40.71μg/ml). Molecular docking of the isatin hybrids against DNA gyrase and quorum-sensing receptor CviR validated the observed in vitro findings. The befitting pharmacokinetic profile of the synthesized drug candidates was ascertained through absorption, distribution, metabolism, excretion and toxicity screening. Conclusion: The remarkable biocompetence of the synthesized triazinoindoles may help to combat drug-resistant infections.

Machine learning-assisted optimized production of quorum quenching anthraquinones in rhubarb

BackgroundThis first attempt study focused on assessing the quorum quenching (QQ) capabilities of four keystone anthraquinones—chrysophanol, emodin, aloe emodin, and rhein— and their analogs found in the medicinal rhubarb herb. Virtual screening was utilized to determine the affinity of these compounds against quorum-sensing receptors. The anthraquinone with the highest binding score will be prioritized for optimized production via machine learning. Methods45 anthraquinone compounds and two quorum sensing receptors, SdiA(AHL type) and LsrB(AI-2 type) were obtained for virtual screening. An optimized random forest with bootstrap resampling was used for feature importance to see how parameters such as concentrations and k constants influence chrysophanol production. Significant findingsVirtual screening methods reveal that chrysophanol compounds could not only demonstrate bioenergy-simulating and electron-shuttling capabilities from prior studies but also have the potential to inhibit bacterial communication, through quorum quenching. It was observed that chrysophanol 8-(6-galloylglucoside) and chrysophanol 8-(6-O-galloyl-beta-D-glucopyranoside) have the highest QQ potential with an affinity of −12.508 and −10.133, respectively. Random forest revealed the importance of high consumption of reactant and the influence of rhein and aloe emodin in the production of chrysophanol. Inhibition of hydroxylase while upregulation of oxidase could potentially lead to higher chrysophanol yield.

Mitigation of quorum sensing mediated virulence factors of Pseudomonas aeruginosa: the role of Meldrum's acid activated furan.

The rapid emergence of drug resistant pathogens is a major threat which has warranted the development of alternative strategies to combat infectious diseases. In this work, we have tested the anti-virulent activity of Meldrum's acid activated furan (MAF) and 1,3-dimethyl barbituric acid activated furan (BAF) against Chromobacterium violaceum and Pseudomonas aeruginosa. It was found that MAF significantly reduced the violacein production and biofilm formation of C. violaceum at sub-inhibitory concentrations. The quorum sensing (QS) regulated virulence factors of P. aeruginosa including biofilm formation, motility, pigment production, and elastase activity were also found to be reduced considerably at sub-inhibitory concentrations of MAF. Additionally, MAF downregulated the expression of genes in the QS circuitry of P. aeruginosa, demonstrating the potential of MAF in lowering the pathogenicity of P. aeruginosa. In silico studies demonstrated the potential of MAF to compete with the signaling molecules of C. violaceum and P. aeruginosa for the QS receptor interaction. In vivo studies using Caenorhabditis elegans demonstrated the anti-pathogenicity of MAF by enhancing the survival of P. aeruginosa-infected C. elegans. These results suggest that activated furan compounds could be potential inhibitors of QS-mediated virulence factors in C. violaceum and P. aeruginosa, encouraging their use in combating multidrug-resistant pathogens.

Marine-Derived Furanones Targeting Quorum-Sensing Receptors in Pseudomonas aeruginosa: Molecular Insights and Potential Mechanisms of Inhibition.

The quorum-sensing (QS) machinery in disease-causing microorganisms is critical in developing antibiotic resistance. In Pseudomonas aeruginosa, QS is involved in biofilm formation, virulence factors production, and general tolerance to antimicrobials. Owing to the major role QS plays, interference in the process is probably a facile route to overcome antimicrobial resistance. Some furanone-derived compounds from marine sources have shown promising anti-QS activity. However, their protein targets and potential mechanisms of action have not been explored. To elucidate their potential protein targets in this study, marine metabolites with furanone backbones similar to their cognitive autoinducers (AIs) were screened against various QS receptors (LasR, RhlR, and PqsR) using molecular docking and molecular dynamics (MD) simulation techniques. The order by which the compounds bind to the receptors follows LasR > RhlR > PqsR. Compounds exhibited remarkable stability against LasR and RhlR, likely because the AIs of these receptors are structural analogs of furanones. Furanones with shorter alkyl side chains bound strongly against RhlR. The presence of halogens improved binding against various receptors. PqsR, with its hydrophobic-binding site and structurally different AIs, showed weaker binding. This study provides a molecular basis for the design of potent antagonists against QS receptors using marine-derived furanones.

Molecular Docking Approach Reveals The Potential Role Of Psidium Guajava Leaf Extract Compounds As Quorum-Sensing Inhibitors Targeting Pseudomonas Aeruginosa’s Lasr

Quorum-sensing LasR antagonism is evolving as a primary focus in promising new antivirulence approaches for treating bacterial infections. Pseudomonas aeruginosa’s LasR is a target receptor for developing alternative medicines in chronic wounds because it acts as an autoinducer in biofilm formation. Our research aims to predict the biofunction of Psidium guajava leaf water extract as an inhibitor of the quorum-sensing LasR of Pseudomonas aeruginosa. The 3D structures of five bioactive compounds (quercetin, gallocatechin, esculin, 3-sinapoylquinic acid, ellagic acid) and N-3-Oxo-Dodecanoyl-L-Homoserine Lactone (as positive control) were obtained from the PubChem Database. The Protein Data Bank database is used to download LasR. The active site of the LasR protein was determined using Molegro Virtual Docker 5.0. The docking simulation uses Molegro Virtual Docker 5.0 and Discovery Studio program version 21.1.1 for visualization. The results showed that the five compounds could bind to LasR at the active site and substrate binding, leading to the compound’s potential as an antibacterial for Pseudomonas aeruginosa by inhibiting the quorum-sensing receptor LasR. The 3-sinapoylquinic acid-LasR complex shows the lowest binding energy, namely -311.2 kJ/mol.

Antimicrobial and anti-virulence activities of 4-shogaol from grains of paradise against gram-negative bacteria: Integration of experimental and computational methods

Ethnopharmacological relevanceBacterial resistance to antibiotics is a growing global concern, highlighting the urgent need for new antimicrobial candidates. Aframomum melegueta was traditionally used for combating urinary tract and soft tissue infections, which implies its potential as an antimicrobial agent. Aim of studyThis study was designed to explore the antibacterial and anti-virulence capabilities of 4-shogaol isolated from A. melegueta seeds versus gram-negative bacteria: Serratia marcescens, Klebsiella pneumoniae, Acinetobacter baumannii, and the clinically important pathogen Pseudomonas aeruginosa. Materials and methods4-Shogeol was isolated from A. melegueta seeds and its MICs were determined for Acinetobacter baumannii (ATCC-17978), Pseudomonas aeruginosa (ATCC-27853), Klebsiella pneumoniae (ATCC-700603), and Serratia marcescens clinical isolate. The anti-efflux activity and effect on the bacterial cell membrane for the compound were evaluated. Furthermore, the anti-virulence activities of the compound were evaluated. The effects of 4-shogeol at sub-MIC on bacterial motility, biofilm formation, and production of virulent enzymes and pigments were assessed. The anti-quorum sensing activities of 4-shogeol were evaluated virtually and by quantification its effect on the expression of quorum sensing encoding genes. The in vivo protection assay was conducted to evaluate the effect of 4-shogaol on the P. aeruginosa capacity to induce pathogenesis in mice. Finally, the effect of shogaol-antibiotics combination was assessed. ResultsThe research revealed that 4-shogaol's antibacterial action primarily involves disrupting the bacterial cell membrane and efflux pumps. It also exhibited significant anti-virulence effects by reducing biofilm development and repressing virulence factors production, effectively protecting mice against P. aeruginosa infection. Furthermore, when combined with antibiotics, 4-shogaol demonstrated synergistic effects, leading to reduced minimum inhibitory concentrations (MICs) against P. aeruginosa. Its anti-virulence properties were linked to its ability to disrupt bacterial quorum sensing (QS) mechanisms, as evidenced by its interaction with QS receptors and downregulation of QS-related genes. Notably, in silico analysis indicated that 4-shogaol exhibited strong binding affinity to different P. aeruginosa QS targets. ConclusionThese findings suggest that 4-shogaol holds promise as an effective anti-virulence agent that can be utilized in combination with antibiotics for treating severe infections caused by gram-positive bacteria.

Promoter selectivity of the RhlR quorum-sensing transcription factor receptor in Pseudomonas aeruginosa is coordinated by distinct and overlapping dependencies on C4-homoserine lactone and PqsE.

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-β-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa.

Repurposing albendazole as a potent inhibitor of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa: Novel prospects of a classical drug

Pseudomonas aeruginosa has emerged as a critical superbug that poses a serious threat to public health. Owing to its virulence and multidrug resistance profiles, the pathogen demands immediate attention for devising alternate intervention strategies. In an attempt to repurpose drugs against P. aeruginosa, this preclinical study was aimed at investigating the antivirulence prospects of albendazole (AbZ), an FDA-approved anti-helminthic drug, recently predicted to disrupt quorum sensing (QS) in Chromobacterium violaceum. AbZ was scrutinized for its quorum quenching (QQ) prospects, effect on bacterial virulence, different motility phenotypes, and biofilm formation in vitro. Additionally, in silico analysis was employed to predict the molecular interactions between AbZ and QS receptors. At sub-inhibitory levels, AbZ demonstrated anti-QS activity and significantly abrogated AHL biosynthesis in P. aeruginosa. Moreover, AbZ significantly downregulated the transcript levels of QS- (lasI/lasR, rhlI/rhlR, and pqsA/pqsR) and QS-dependent virulence (aprA, lasA, lasB, plcH, and toxA) genes in P. aeruginosa. This coincided with reduced hemolysin, alginate, pyocyanin, rhamnolipids, total protease, and elastase production, thereby lowering phenotypic virulence. Molecular docking with AbZ further revealed strong associations and high binding energies with LasR (−8.8 kcal/mol), RhlR (−6.5 kcal/mol), and PqsR (−6.3 kcal/mol) receptors. AbZ also impeded bacterial motility and abolished EPS production, severely compromising pseudomonal biofilm formation. For the first time, AbZ was shown to interfere with QS circuitry and consequently disarming pseudomonal virulence. Hence, AbZ can be exploited for its antivirulence properties against P. aeruginosa.

The Anti-Virulence Activities of the Antihypertensive Drug Propranolol in Light of Its Anti-Quorum Sensing Effects against Pseudomonas aeruginosa and Serratia marcescens.

The development of bacterial resistance is an increasing global concern that requires discovering new antibacterial agents and strategies. Bacterial quorum sensing (QS) systems play important roles in controlling bacterial virulence, and their targeting could lead to diminishing bacterial pathogenesis. In this context, targeting QS systems without significant influence on bacterial growth is assumed as a promising strategy to overcome resistance development. This study aimed at evaluating the anti-QS and anti-virulence activities of the β-adrenoreceptor antagonist propranolol at sub-minimal inhibitory concentrations (sub-MIC) against two Gram-negative bacterial models Pseudomonas aeruginosa and Serratia marcescens. The effect of propranolol on the expression of QS-encoding genes was evaluated. Additionally, the affinity of propranolol to QS receptors was virtually attested. The influence of propranolol at sub-MIC on biofilm formation, motility, and production of virulent factors was conducted. The outcomes of the propranolol combination with different antibiotics were assessed. Finally, the in vivo protection assay in mice was performed to assess propranolol's effect on lessening the bacterial pathogenesis. The current findings emphasized the significant ability of propranolol at sub-MIC to reduce the formation of biofilms, motility, and production of virulence factors. In addition, propranolol at sub-MIC decreased the capacity of tested bacteria to induce pathogenesis in mice. Furthermore, propranolol significantly downregulated the QS-encoding genes and showed significant affinity to QS receptors. Finally, propranolol at sub-MIC synergistically decreased the MICs of different antibiotics against tested bacteria. In conclusion, propranolol might serve as a plausible adjuvant therapy with antibiotics for the treatment of serious bacterial infections after further pharmacological and pharmaceutical studies.