QS-regulated Genes Research Articles

Repurposing promethazine hydrochloride to inhibit biofilm formation against Burkholderia thailandensis.

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, an intracellular pathogen with a high mortality rate and significant antibiotic resistance. The high mortality rate and resistance to antibiotics have drawn considerable attention from researchers studying melioidosis. This study evaluated the effects of various concentrations (75, 50, and 25µg/mL) of promethazine hydrochloride (PTZ), a potent antihistamine, on biofilm formation and lipase activity after 24h of exposure to B. thailandensis E264. A concentration-dependent decrease in both biofilm biomass and lipase activity was observed. RT-PCR analysis revealed that PTZ treatment not only made the biofilm structure loose but also reduced the expression of btaR1, btaR2, btaR3, and scmR. Single gene knockouts of quorum sensing (QS) receptor proteins (∆btaR1, ∆btaR2, and ∆btaR3) were successfully constructed. Deletion of btaR1 affected biofilm formation in B. thailandensis, while deletion of btaR2 and btaR3 led to reduced lipase activity. Molecular docking and biological performance results demonstrated that PTZ inhibits biofilm formation and lipase activity by suppressing the expression of QS-regulated genes. This study found that repositioning PTZ reduced biofilm formation in B. thailandensis E264, suggesting a potential new approach for combating melioidosis.

Phenotypic memory in quorum sensing.

Quorum sensing (QS) is a regulatory mechanism used by bacteria to coordinate group behavior in response to high cell densities. During QS, cells monitor the concentration of external signals, known as autoinducers, as a proxy for cell density. QS often involves positive feedback loops, leading to the upregulation of genes associated with QS signal production and detection. This results in distinct steady-state concentrations of QS-related molecules in QS-ON and QS-OFF states. Due to the slow decay rates of biomolecules such as proteins, even after removal of the initial stimuli, cells can retain elevated levels of QS-associated biomolecules for extended periods of time. This persistence of biomolecules after the removal of the initial stimuli has the potential to impact the response to future stimuli, indicating a memory of past exposure. This phenomenon, which is a consequence of the carry-over of biomolecules rather than genetic inheritance, is known as "phenotypic" memory. This theoretical study aims to investigate the presence of phenotypic memory in QS and the conditions that influence this memory. Numerical simulations based on ordinary differential equations and analytical modeling were used to study gene expression in response to sudden changes in cell density and extracellular signal concentrations. The model examined the effect of various cellular parameters on the strength of QS memory and the impact on gene regulatory dynamics. The findings revealed that QS memory has a transient effect on the expression of QS-responsive genes. These consequences of QS memory depend strongly on how cell density was perturbed, as well as various cellular parameters, including the Fold Change in the expression of QS-regulated genes, the autoinducer synthesis rate, the autoinducer threshold required for activation, and the cell growth rate.

Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump.

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were re-synthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing, revealing a strong inhibitor of QS-regulated genes, and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump, thereby inhibiting the autogenerated positive feedback and its enhanced signal-molecule synthesis.

High-Throughput, Quantitative Screening of Quorum-Sensing Inhibitors Based on a Bacterial Biosensor.

Quorum sensing (QS) is a cell-cell communication mechanism by which bacteria synchronize social behaviors such as biofilm formation and virulence factor secretion by producing and sensing small molecular signals. Quorum quenching (QQ) by degrading signals or blocking signal transmissions has become a promising strategy for disrupting QS and preventing bacterial infection and biofilm formation. However, studies of high-throughput screening and identification approaches for quorum-sensing inhibitors (QSIs) are still inadequate. In this work, we developed a sensitive, high-throughput approach for screening QSIs based on the bacterial biosensor strain Agrobacterium tumefaciens N5 (pBA7P), which contains a traG gene promoter induced by QS signals fused with a promoterless β-lactamase gene reporter. Using this approach, we identified 31 QQ bacteria from ∼2000 soil bacterial isolates, some belonging to the genera Bosea, Cupriavidus, and Flavobacterium that have not been reported previously as QQ bacteria. We also identified four QS inhibitory compounds and one QS signal analogue from ∼5000 small-molecule compounds, which profoundly affected the expression of QS-regulated genes and phenotypes of the pathogenic bacteria. This high-throughput screening system is effective and sensitive for screening of both QQ microbes and small molecules, enabling the discovery of a wide variety of biocompatible compounds.

Cyclopropane-Containing Specialized Metabolites from the Marine Cyanobacterium cf. Lyngbya sp.

Marine cyanobacteria are known to produce structurally diverse bioactive specialized metabolites during bloom occurrence. These ecologically active allelochemicals confer chemical defense for the microalgae from competing microbes and herbivores. From a collection of a marine cyanobacterium, cf. Lyngbya sp., a small quantity of a new cyclopropane-containing molecule, benderadiene (2), and lyngbyoic acid (1) were purified and characterized using spectroscopic methods. Using live reporter quorum-sensing (QS) inhibitory assays, based on P. aeruginosa PAO1 lasB-gfp and rhlA-gfp strains, both compounds were found to inhibit QS-regulated gene expression in a dose-dependent manner. In addition to lyngbyoic acid being more active in the PAO1 lasB-gfp biosensor strain (IC50 of 20.4 µM), it displayed anti-biofilm activity when incubated with wild-type P. aeruginosa. The discovery of lyngbyoic acid in relatively high amounts provided insights into its ecological significance as a defensive allelochemical in targeting competing microbes through interference with their QS systems and starting material to produce other related analogs. Similar strategies could be adopted by other marine cyanobacterial strains where the high production of other lipid acids has been reported. Preliminary evidence is provided from the virtual molecular docking of these cyanobacterial free acids at the ligand-binding site of the P. aeruginosa LasR transcriptional protein.

Novel quorum sensing inhibitor Echinatin as an antibacterial synergist against Escherichia coli.

A new antibacterial strategy based on inhibiting bacterial quorum sensing (QS) has emerged as a promising method of attenuating bacterial pathogenicity and preventing bacterial resistance to antibiotics. In this study, we screened Echinatin (Ech) with high-efficiency anti-QS from 13 flavonoids through the AI-2 bioluminescence assay. Additionally, crystal violet (CV) staining combined with confocal laser scanning microscopy (CLSM) was used to evaluate the effect of anti-biofilm against Escherichia coli (E. coli). Further, the antibacterial synergistic effect of Ech and marketed antibiotics were measured by broth dilution and Alamar Blue Assay. It was found that Ech interfered with the phenotype of QS, including biofilm formation, exopolysaccharide (EPS) production, and motility, without affecting bacterial growth and metabolic activity. Moreover, qRT-PCR exhibited that Ech significantly reduced the expression of QS-regulated genes (luxS, pfs, lsrB, lsrK, lsrR, flhC, flhD, fliC, csgD, and stx2). More important, Ech with currently marketed colistin antibiotics (including colistin B and colistin E) showed significantly synergistically increased antibacterial activity in overcoming antibiotic resistance of E. coli. In summary, these results suggested the potent anti-QS and novel antibacterial synergist candidate of Ech for treating E. coli infections.

Superior inhibition of virulence and biofilm formation of Pseudomonas aeruginosa PAO1 by phyto-synthesized silver nanoparticles through anti-quorum sensing activity.

Quorum sensing (QS)-regulated bacterial biofilm formation is a crucial issue in causing resistance against existing antibiotics. There is a considerable necessity to disrupt the interrelationship between bacterial QS, virulence, and biofilm formation. Disabling QS could be a novel tactic of great clinical importance. Here, we biosynthesized silver nanoparticles (Ka-AgNPs) using the aqueous leaf extract of Koelreuteria paniculata as a reducing and capping agents. The UV-Vis spectroscopy confirmed the synthesis of Ka-AgNPs as a characterization peak observed at 420nm. TEM image revealed the spherical shape distribution of Ka-AgNPs with average particle size of 30.0±5nm. The anti-QS activity of Ka-AgNPs was tested against a bio-indicator bacterium Chromobacterium violaceum 12472 and a multi-drug resistant model strain of Pseudomonas aeruginosa (PAO1). The results demonstrated that the Ka-AgNPs superiorly inhibited QS-regulated virulence factors in PAO1 without affecting cell viability compared to chemically synthesized AgNPs (Cs-AgNPs). The Ka-AgNPs effectively suppressed the formation of biofilm of PAO1. RT-PCR results revealed that the Ka-AgNPs inhibited the expression of QS-regulated virulence genes of PAO1. These results suggest that the phyto-synthesized AgNPs could be used as promising anti-infective agents for treating drug-resistant P. aeruginosa.

The effects of LL-37 on virulence factors related to the quorum sensing system of Pseudomonas aeruginosa.

BackgroundAntimicrobial peptides (AMPs) have shown promise in the treatment of multi-resistant pathogens. It was therefore of interest to analyze the effects of the AMP LL-37 on the regulation of several virulence factors related to the quorum sensing (QS) system of Pseudomonas aeruginosa (P. aeruginosa) in vitro.MethodsThe minimum inhibitory concentration (MIC) was evaluated by the micro broth dilution method. The expression of QS-related and QS-regulated virulence factor genes was also evaluated. Exotoxin A activity was measured with the nicotinamide adenine dinucleotide (NAD) (Coenzyme I) method; Elastase activity was detected with the elastin-Congo red (ECR) method; Pyocyanin detection was performed using the chloroform extraction method. The effects of LL-37 were assessed by measuring the expression changes of the virulence protein-encoding genes of the strains with quantitative polymerase chain reaction (PCR).ResultsThe MIC of LL-37 against both P. aeruginosa reference strain (ATCC 15692 PAO1) and PA-ΔlasI/rhII was therefore determined to be 256 µg/mL. LL-37 at sub-minimum inhibitory concentrations (sub-MICs) had no significant effects on P. aeruginosa bacterial growth (P>0.05), but significantly downregulated the expression of all 3 virulence factors.ConclusionsInterestingly, this effect appeared to be dose-related. These findings suggest that LL-37 could be a potential candidate for QS inhibition against bacterial infection and may have significant clinical potential in the treatment of P. aeruginosa biofilms.

Phloretin, an Apple Phytoalexin, Affects the Virulence and Fitness of Pectobacterium brasiliense by Interfering With Quorum-Sensing

The effects of phloretin a phytoalexin from apple, was tested on Pectobacterium brasiliense (Pb1692), an emerging soft-rot pathogen of potato. Exposure of Pb1692 to 0.2 mM phloretin a concentration that does not affect growth, or to 0.4 mM a 50% growth inhibiting concentration (50% MIC), reduced motility, biofilm formation, secretion of plant cell wall-degrading enzymes, production of acyl–homoserine lactone (AHL) signaling molecules and infection, phenotypes that are associated with bacterial population density-dependent system known as quorum sensing (QS). To analyze the effect of growth inhibition on QS, the activity of ciprofloxacin, an antibiotic that impairs cell division, was compared to that of phloretin at 50% MIC. Unlike phloretin, the antibiotic hardly affected the tested phenotypes. The use of DH5α, a QS-negative Escherichia coli strain, transformed with an AHL synthase (ExpI) from Pb1692, allowed to validate direct inhibition of AHL production by phloretin, as demonstrated by two biosensor strains, Chromobacterium violaceaum (CV026) and E. coli (pSB401). Expression analysis of virulence-related genes revealed downregulation of QS-regulated genes (expI, expR, luxS, rsmB), plant cell wall degrading enzymes genes (pel, peh and prt) and motility genes (motA, fim, fliA, flhC and flhD) following exposure to both phloretin concentrations. The results support the inhibition of ExpI activity by phloretin. Docking simulations were used to predict the molecular associations between phloretin and the active site of ExpI, to suggest a likely mode of action for the compound’s inhibition of virulence.

Effect of Titanium Dioxide Nanoparticles on the Expression of Efflux Pump and Quorum-Sensing Genes in MDR Pseudomonas aeruginosa Isolates.

Most of the infections caused by multi-drug resistant (MDR) P. aeruginosa strains are extremely difficult to be treated with conventional antibiotics. Biofilm formation and efflux pumps are recognized as the major antibiotic resistance mechanisms in MDR P. aeruginosa. Biofilm formation by P. aeruginosa depends mainly on the cell-to-cell communication quorum-sensing (QS) systems. Titanium dioxide nanoparticles (TDN) have been used as antimicrobial agents against several microorganisms but have not been reported as an anti-QS agent. This study aims to evaluate the impact of titanium dioxide nanoparticles (TDN) on QS and efflux pump genes expression in MDR P. aeruginosa isolates. The antimicrobial susceptibility of 25 P. aeruginosa isolates were performed by Kirby–Bauer disc diffusion. Titanium dioxide nanoparticles (TDN) were prepared by the sol gel method and characterized by different techniques (DLS, HR-TEM, XRD, and FTIR). The expression of efflux pumps in the MDR isolates was detected by the determination of MICs of different antibiotics in the presence and absence of carbonyl cyanide m-chlorophenylhydrazone (CCCP). Biofilm formation and the antibiofilm activity of TDN were determined using the tissue culture plate method. The effects of TDN on the expression of QS genes and efflux pump genes were tested using real-time polymerase chain reaction (RT-PCR). The average size of the TDNs was 64.77 nm. It was found that TDN showed a significant reduction in biofilm formation (96%) and represented superior antibacterial activity against P. aeruginosa strains in comparison to titanium dioxide powder. In addition, the use of TDN alone or in combination with antibiotics resulted in significant downregulation of the efflux pump genes (MexY, MexB, MexA) and QS-regulated genes (lasR, lasI, rhll, rhlR, pqsA, pqsR) in comparison to the untreated isolate. TDN can increase the therapeutic efficacy of traditional antibiotics by affecting efflux pump expression and quorum-sensing genes controlling biofilm production.

Effects of Gentamicin-Loaded Chitosan-ZnO Nanocomposite on Quorum-Sensing Regulation of Pseudomonas Aeruginosa.

Cell density-based intercellular signaling mechanism is known as Quorum sensing (QS); it serves a significant role in regulating the pathogenic factors. The objective of the present study was to assess the influence of chitosan-zinc oxide nanocomposite (CH-ZnO nanocomposite), alone and in combination with gentamicin, on the sensitivity to hydrogen peroxide (H2O2), the production of pathogenic factors and QS-regulated genes of Pseudomonas aeruginosa. The efficacy of the minimum inhibitory concentration (MIC) and 1/4 MIC of the CH-ZnO nanocomposite, alone and in combination with gentamicin, on the sensitivity to H2O2, pyocyanin secretion, swarming and twitching motilities was evaluated. In addition, the expression of some QS-regulated genes including rhlI, rhlR, lasI and lasR genes was measured by Real-time quantitative PCR (RT-qPCR) following exposure to the nanocomposite. The results demonstrated that at MIC concentrations, the gentamicin-loaded CH-ZnO nanocomposite significantly inhibited QS-regulated phenotypes such as pyocyanin secretion (82.4%), swarming (76%) and twitching (73.6%) motilities; further it increased the inhibition growth zone (134.5%), as well as, at 1/4 MIC concentration decreased the expression of lasI (72%), lasR (78%), rhlI (76%) and rhlR (82%) genes; as compared to untreated P. aeruginosa PAO1 (P < 0.05). Our results also demonstrated that the CH-ZnO nanocomposite combined with gentamicin could be a potential innovative candidate, which could be broadly applied in the treatment of P. aeruginosa infections.

EGCG-Mediated Potential Inhibition of Biofilm Development and Quorum Sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa), one of the dangerous multidrug resistance pathogens, orchestrates virulence factors production through quorum sensing (QS). Since the exploration of QS inhibitors, targeting virulence to circumvent bacterial pathogenesis without causing significant growth inhibition is a promising approach to treat P. aeruginosa infections. The present study has evaluated the anti-QS and anti-infective activity of epigallocatechin-3-gallate (EGCG), a bioactive ingredient of the traditional green tea, against P. aeruginosa. EGCG showed significant inhibitory effects on the development of biofilm, protease, elastase activity, swimming, and swarming motility, which was positively related to the production of C4-AHL. The expression of QS-related and QS-regulated virulence factors genes was also evaluated. Quantitative PCR analysis showed that EGCG significantly reduced the expression of las, rhl, and PQS genes and was highly correlated with the alterations of C4-AHL production. In-vivo experiments demonstrated that EGCG treatment reduced P. aeruginosa pathogenicity in Caenorhabditis elegans (C. elegans). EGCG increased the survival of C. elegans by 23.25%, 30.04%, and 36.35% in a dose-dependent manner. The findings of this study strongly suggest that EGCG could be a potential candidate for QS inhibition as an anti-virulence compound against bacterial infection.

Antibiotic intervention redisposes bacterial interspecific interacting dynamics in competitive environments.

Interspecific interaction happens frequently among bacterial species and can promote the colonization of polymicrobial community in various environments. However, it is not clear whether the intervention of antibiotics, which is a common therapeutic method for infectious disease, will influence the interacting dynamics of different pathogenic bacteria. By using the frequently co-isolated bacteria Pseudomonas aeruginosa and Staphylococcus aureus as models, here we identify an antibiotic-determined mutual invasion relationship between bacterial pathogens. We show that although P. aeruginosa has a significant intrinsic competitive advantage over S. aureus by producing the quorum-sensing (QS)-controlled anti-staphylococcal molecules, methicillin-resistant S. aureus (MRSA) can inhibit neighbouring P. aeruginosa in the presence of subinhibitory aminoglycoside antibiotics (e.g. streptomycin) to P. aeruginosa. Importantly, subinhibitory streptomycin decreases the expression of QS-regulated genes in P. aeruginosa and thus relieves the survival stress of MRSA brought by P. aeruginosa. On the other side, the iron-uptake systems and pathogenicity of MRSA can be enhanced by the extracellular products of streptomycin-treated P. aeruginosa. Therefore, this study provides an explanation for the substitution of dominant species and persistent coexistence of bacterial pathogens in the host with repeated antibiotic therapies and contributes to further understanding the pathogenesis of chronic polymicrobial infections.

Transcriptome analysis expands the potential roles of quorum sensing in biodegradation and physiological responses to microcystin

Bacterial degradation is one of the most efficient ways to remove microcystins (MCs), the most frequently detected toxin in cyanobacterial blooms. Using Novosphingobium sp. ERW19 as a representative strain, our laboratory previously demonstrated that quorum sensing (QS), the cell density-dependent gene regulation system, positively regulates biodegradation of MCs via transcriptional activation of mlr-pathway-associated genes. Increasing evidence indicates that QS is involved in a wide spectrum of regulatory circuits, but it remains unclear which physiological processes in MC degradation besides the expression of MC-degrading genes are also subject to QS-dependent regulation. This study used transcriptome analysis to identify QS-regulated genes during degradation of MCs. A large percentage (up to 32.6%) of the genome of the MC-degrading bacterial strain Novosphingobium sp. ERW19 was significantly differentially expressed in the corresponding QS mutants. Pathway enrichment analysis of QS-regulated genes revealed that QS mainly influenced metabolism-associated pathways, particularly those related to amino acid metabolism, carbohydrate metabolism, and biodegradation and metabolism of xenobiotics. In-depth functional interpretation of QS-regulated genes indicated a variety of pathways were potentially associated with bacterial degradation or physiological responses to MCs, including genes involved in cell motility, cytochrome P450-dependent metabolism of xenobiotics, glutathione S-transferase (GST), envelope stress response, and ribosomes. Furthermore, QS may be involved in regulating the initial and final steps of the catabolic pathway of phenylacetic acid, an intermediate product of MC degradation. Collectively, this global survey of QS-regulated genes in a MC-degrading bacterial strain facilitates a deeper understanding of QS-controlled processes that may be important for bacterial degradation of MCs or may contribute to the physiological responses of bacteria to MCs.

New insights into the antibacterial and quorum sensing inhibition mechanism of Artemisia argyi leaf extracts towards Pseudomonas aeruginosa PAO1.

The online version contains supplementary material available at 10.1007/s13205-021-02663-5.

Traditional Chinese Medicine Tanreqing Inhibits Quorum Sensing Systems in Pseudomonas aeruginosa.

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.

During bacteremia, Pseudomonas aeruginosa PAO1 adapts by altering the expression of numerous virulence genes including those involved in quorum sensing.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that produces numerous virulence factors and causes serious infections in trauma patients and patients with severe burns. We previously showed that the growth of P. aeruginosa in blood from severely burned or trauma patients altered the expression of numerous genes. However, the specific influence of whole blood from healthy volunteers on P. aeruginosa gene expression is not known. Transcriptome analysis of P. aeruginosa grown for 4 h in blood from healthy volunteers compared to that when grown in laboratory medium revealed that the expression of 1085 genes was significantly altered. Quorum sensing (QS), QS-related, and pyochelin synthesis genes were downregulated, while genes of the type III secretion system and those for pyoverdine synthesis were upregulated. The observed effect on the QS and QS-related genes was shown to reside within serum fraction: growth of PAO1 in the presence of 10% human serum from healthy volunteers significantly reduced the expression of QS and QS-regulated genes at 2 and 4 h of growth but significantly enhanced their expression at 8 h. Additionally, the production of QS-regulated virulence factors, including LasA and pyocyanin, was also influenced by the presence of human serum. Serum fractionation experiments revealed that part of the observed effect resides within the serum fraction containing <10-kDa proteins. Growth in serum reduced the production of many PAO1 outer membrane proteins but enhanced the production of others including OprF, a protein previously shown to play a role in the regulation of QS gene expression. These results suggest that factor(s) within human serum: 1) impact P. aeruginosa pathogenesis by influencing the expression of different genes; 2) differentially regulate the expression of QS and QS-related genes in a growth phase- or time-dependent mechanism; and 3) manipulate the production of P. aeruginosa outer membrane proteins.

Quorum quenching by 2-Hydroxyanisole extracted from Solanum torvum on Pseudomonas aeruginosa and its inhibitory action upon LasR protein

Among the list of the most dangerous superbugs, Pseudomonas aeruginosa ranks top of the table with its deadly characteristics of multi-drug resistance and ability to form biofilm during opportunistic infections to patients those are immune-compromised. MDX and XDR of this strain urged researchers to reveal better antibacterial compounds. Solanum torvum (commonly called as pea eggplant) is well known for its medicinal values however, the roots of this plant had less attention in pharmacological studies. The objective of this study is twofold. Firstly, to compare the interactions exhibited between2-Hydroxyanisole and odDHL with LasRprotein. Secondly, to explore the impact of molecule of interest on genes related to vital pathways. Three-dimensional docking studies for the two ligands were performed using surflex-dock package - Sybyl-x 2.0 whereas differentially regulated genes were identified by volcano plot, heat map prediction and KEGG pathway. It was predicted that 2-Hydroxyanisole may form three hydrogen bonds with LasR protein and revealed a decreased level of gene expression (%).After treatment with Solanum torvum Root Extract (STRE),a total of 26,418,456 reads of P. aeuroginosa were mapped to various pathways, of which, QS-regulated genes such as LasR, coIII (coxB), fliD genes along with 104 genes were down regulated. The high C Score calculated for 2-Hydroxyanisole and altered gene expression upon STRE treatment pave the pathway for designing the future in vitro experiments to validate these inhibitory effects thereby exploring the underlying mechanisms of action of 2-Hydroxyanisole to restrict the virulence of P. aeruginosa.

Repurposing anti-diabetic drug "Sitagliptin" as a novel virulence attenuating agent in Serratia marcescens.

BackgroundSerratia marcescens is an emerging pathogen that causes a variety of health care associated infections. S. marcescens is equipped with an arsenal of virulence factors such as biofilm formation, swimming and swarming motilities, prodigiosin, protease and others which enable it to initiate and cause the infection. These virulence factors are orchestrated under the umbrella of an intercellular communication system named Quorum sensing (QS). QS allows bacterial population to synchronize the expression of virulence genes upon detection of a chemical signaling molecule. Targeting bacterial virulence is a promising approach to attenuate bacteria and enhances the ability of immune system to eradicate the bacterial infection. Drug repurposing is an advantageous strategy that confers new applications for drugs outside the scope of their original medical use. This promising strategy offers the use of safe approved compounds, which potentially lowers the costs and shortens the time than that needed for development of new drugs. Sitagliptin is dipeptidyl peptidase-4 (DPP-4) inhibitor, is used to treat diabetes mellitus type II as it increases the production of insulin and decreasing the production of glucagon by the pancreas. We aimed in this study to repurpose sitagliptin, investigating the anti-virulence activities of sitagliptin on S. marcescens.MethodsThe effect of sub-inhibitory concentrations of sitagliptin on virulence factors; protease, prodigiosin, biofilm formation, swimming and swarming motilities was estimated phenotypically. The qRT-PCR was used to show the effect of sitagliptin on the expression of QS-regulated virulence genes. The in-vivo protective activity of sitagliptin on S. marcescens pathogenesis was evaluated on mice.ResultsSitagliptin (1 mg/ml) significantly reduced the biofilm formation, swimming and swarming motilities, prodigiosin and protease. The qRT-PCR confirmed the effect on virulence as shown by down regulating the expression of fimA, fimC, flhC, flhD, bsmB, rssB, rsmA, pigP, and shlA genes. Moreover, the in-vivo findings showed the efficient ability of sitagliptin to weaken S. marcescens pathogenesis.ConclusionSitagliptin is a promising anti-virulence agent against S. marcescens that may be beneficial in the control of healthcare associated infections caused by S. marcescens.

Naringenin Inhibition of the Pseudomonas aeruginosa Quorum Sensing Response Is Based on Its Time-Dependent Competition With N-(3-Oxo-dodecanoyl)-L-homoserine Lactone for LasR Binding.

Bacterial quorum sensing (QS) is a cell-to-cell communication system that governs the expression of a large set of genes involved in bacterial–host interactions, including the production of virulence factors. Conversely, the hosts can produce anti-QS compounds to impair virulence of bacterial pathogens. One of these inhibitors is the plant flavonoid naringenin, which impairs the production of QS-regulated Pseudomonas aeruginosa virulence factors. In the present work, we analyze the molecular basis for such inhibition. Our data indicate that naringenin produces its effect by directly binding the QS regulator LasR, hence competing with its physiological activator, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3OC12-HSL). The in vitro analysis of LasR binding to its cognate target DNA showed that the capacity of naringenin to outcompete 3OC12-HSL, when the latter is previously bound to LasR, is low. By using an E. coli LasR-based biosensor strain, which does not produce 3OC12-HSL, we determined that the inhibition of LasR is more efficient when naringenin binds to nascent LasR than when this regulator is already activated through 3OC12-HSL binding. According to these findings, at early exponential growth phase, when the amount of 3OC12-HSL is low, naringenin should proficiently inhibit the P. aeruginosa QS response, whereas at later stages of growth, once 3OC12-HSL concentration reaches a threshold enough for binding LasR, naringenin would not efficiently inhibit the QS response. To test this hypothesis, we analyze the potential effect of naringenin over the QS response by adding naringenin to P. aeruginosa cultures at either time zero (early inhibition) or at stationary growth phase (late inhibition). In early inhibitory conditions, naringenin inhibited the expression of QS-regulated genes, as well as the production of the QS-regulated virulence factors, pyocyanin and elastase. Nevertheless, in late inhibitory conditions, the P. aeruginosa QS response was not inhibited by naringenin. Therefore, this time-dependent inhibition may compromise the efficiency of this flavonoid, which will be effective just when used against bacterial populations presenting low cellular densities, and highlight the importance of searching for QS inhibitors whose mechanism of action does not depend on the QS status of the population.