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Plumbagin inhibits quorum sensing-regulated virulence and biofilms of Gram-negative bacteria: in vitro and in silico investigations
Abstract
The global rise in antimicrobial resistance and lack of discovery of new antimicrobials have created serious concerns. Targeting quorum sensing (QS) and biofilms of pathogenic bacteria is considered a promising approach in antimicrobial drug discovery. This study explored the inhibitory effect of plumbagin against biofilms and QS of Chromobacterium violaceum, Serratia marcescens and Pseudomonas aeruginosa. Violacein production in C. violaceum 12472 was reduced by >80%. The virulent traits of P. aeruginosa PAO1 such as pyocyanin, rhamnolipid and proteases were also inhibited at sub-minimum inhibitory concentrations. Moreover, the biofilms of the test bacteria were reduced by 56–70%. Plumbagin reduced the bacterial adherence and colonization on solid surface. Computational studies gave closer insights regarding the possible modes of action. Molecular dynamics simulations revealed that the protein complexes were quite stable under physiological conditions. This study provides both experimental and computational evidence regarding the efficacy of plumbagin against biofilms and the QS-controlled virulence factors of Gram-negative bacteria.
... The quantitative inhibition of violacein pigment production in C. violaceum 12472 was tested in broth with standard procedure (Qais, Khan, Ahmad, Husain, Al-kheraif, et al., 2021). Briefly, C. violaceum 12472 was cultured to the log phase and 100 μL was culture taken as inoculum in 10 mL of sterile broth. ...
... Previously, we have shown the ability of many plants and phytocompounds to inhibit the biofilm of Gram Àve bacteria(Qais, Ahmad, Husain, Alomar, Ahmad, et al., 2021;Qais et al., 2019;Qais, Khan, Ahmad, Husain, Al-kheraif, et al., 2021;. The combined quantitative and qualitative data confirm the remarkable potency of CZHF against the biofilm of Gram Àve bacteria. ...
The development of antibiotic resistant microbial pathogens has become a global health threat and a major concern in modern medicine. The problem of antimicrobial resistance (AMR) has majorly arisen due to sub‐judicious use of antibiotics in health care and livestock industry. A slow progress has been made in last two decades in discovery of new antibiotics. A new strategy in combatting AMR is to modulate or disarm the microbes for their virulence and pathogenicity. Plants are considered as promising source for new drugs against AMR pathogens. In this study, fraction‐based screening of the Cinnamomum zeylanicum extract was performed followed by detailed investigation of antiquorum sensing and antibiofilm activities of the most active fraction that is, C. zeylanicum hexane fraction (CZHF). More than 75% reduction in violacein pigment of C. violaceum 12472 was overserved. CZHF successfully modulated the virulence of Pseudomonas aeruginosa PAO1 by 60.46%–78.35%. A similar effect was recorded against Serratia marcescens MTCC 97. A broad‐spectrum inhibition of biofilm development was found in presence of sub‐MICs of CZHF. The colonization of bacteria onto the glass coverslips was remarkably reduced apart from the reduction in exopolymeric substances. Alkaloids and terpenoids were found in CZHF. GC/MS analysis revealed the presence of cinnamaldehyde dimethyl acetal, 2‐propenal, coumarin, and α‐copaene as major phytocompounds. This study provides enough evidence to support potency of C. zeylanicum extract in targeting the virulence of Gram −ve pathogenic bacteria. The plant extract or active compounds can be developed as successful drugs after careful in vivo examination to target microbial infections.
Research Highlights
Hexane fraction of Cinnamomum zeylanicum is active against QS and biofilms.
The broad‐spectrum antibiofilm activity was further confirmed by microscopic analysis.
Dimethyl acetal, 2‐propenal, coumarin, α‐copaene, and so forth are major phytocompounds.
... Plumbagin has shown its potential therapeutic benefits on numerous chronic diseases like breast cancer, nonsmall cell lung cancer, melanoma, ovarian, squamous cell carcinomas, pancreatic cancer, and prostate cancer (Hafeez et al., 2013). Also, plumbagin can regulate Gram-negative bacterial virulence and biofilm formation by inhibiting the quorum-sensing (QS) system (Qais et al., 2021). Therefore, plumbagin, as a component of Chinese herbal medicine, has a broad prospect in the medical treatment of many diseases. ...
... substances, which is extracted from the plants of Plumbago. Research has pointed out that plumbagin can also inhibit virulence and biofilm-related QS of GNB (Qais et al., 2021). Besides, plumbagin combined with gentamicin has a synergistic effect on carbapenem-resistant K. pneumoniae (Chen et al., 2020). ...
The global emergence and spread of multi-drug resistant (MDR) strains is becoming increasingly worrisome due to the overuse of broad-spectrum antibiotics. Colistin, the last resort for treating MDR strains infections, has once again returned to the clinician’s choice. However, with the widespread use of colistin, colistin-resistant gram-negative bacteria (GNB) have subsequently emerged, including colistin-resistant Pseudomonas aeruginosa (COL-R PA). Therefore, available solutions are urgently needed to respond to this situation. Here, we inspiringly found that the combination of plumbagin and colistin had an efficiently inhibitory effect for colistin-resistant P. aeruginosa in vitro through checkerboard assay and time-kill assay. The combinatorial inhibition of biofilm formation was clearly demonstrated by crystal violet staining and scanning electron microscopy (SEM), and this combination can not only inhibited biofilm formation but also eradicated the mature biofilm. Erythrocytes hemolysis test showed that plumbagin has negligible hemolysis ability. In addition, the increased survival rate of Galleria mellonella ( G. mellonella ) larva confirmed this combination as same as effective in vivo . As for the mechanism of this combination, propidium iodide (PI) staining showed colistin combined with plumbagin could significantly change the membrane permeability, thus exerting synergistic antibacterial activity. In conclusion, the combination of plumbagin and colistin shows a prominently synergistic antibacterial effect in vitro and in vivo , providing a promising option for the therapy of COL-R PA infection.
... These advantages can be addressed by modifying the route of administration [27,28]; therefore, natural drugs hold great promise in the treatment of cancer. As a natural quinone, plumbagin exhibits various biological activities, such as antibacterial [29], immunomodulatory [30] and pro-oxidative stress [31] activities. The anti-cancer effects of plumbagin have been shown in pancreatic cancer [32], liver cancer [33], oesophageal cancer [34], gastric cancer [35] and other digestive system tumours. ...
Objective
This study examined the mechanism through which plumbagin induces ferroptosis of colon cancer cells.
Methods
CCK-8 assay was performed to examine the viability of colon cancer cells (SW480 and HCT116 cells) after they were treated with 0-, 5-, 10-, 15- and 20-μmol/L plumbagin. Colony formation assay and Transwell assay were used to examine the effects of 15-μmol/L plumbagin on the proliferation, invasive ability. The ferroptosis of SW480 and HCT116 cells and the expression of p-p53, p53 and SLC7A11 were analysed. The effects of blocking necrosis, apoptosis and ferroptosis on the anti-cancer effects of plumbagin were examined. After p53 was silenced, the effects of plumbagin on proliferation, invasion, ferroptosis and SLC7A11 expression were assessed. A tumour-bearing nude mouse model was used to examine the effects of p53 silencing and/or plumbagin on tumour growth, ferroptosis and SLC7A11 expression.
Results
Plumbagin inhibited the proliferation of SW480 and HCT116 cells and their invasive and colony-forming abilities. It increased Fe²⁺ levels but significantly decreased GSH and GPX4 levels. When ferroptosis was inhibited, the effects of plumbagin on colon cancer cells were significantly alleviated. Plumbagin promoted the expression and phosphorylation of p53 and inhibited the mRNA and protein levels of SLC7A11. Silencing of p53 counteracted the effects of plumbagin on the ferroptosis and biological behaviour of SW480 and HCT116 cells. In mouse models of colon cancer, silencing of p53 attenuated the tumour-suppressing effects of plumbagin as well as its inhibitory effects on the protein level of SLC7A11 and restored the expression of GSH and GPX4.
Conclusion
Plumbagin promotes ferroptosis and inhibits cell proliferation and invasion by decreasing the protein expression of SLC7A11 through p53.
... These effects include antibacterial, antiviral, antineoplastic, antiprotozoal, and antifungal effects [7]. A literature review revealed the presence of biologically active substances that possess aromatic, heteroaromatic, and acyl substituents [8,9]. The biological activity of the parent molecule is known to be enhanced by various substituents, such as benzene, substituted benzene, nitrogen, sulfur, or halogen [10]. ...
Multiple diseases are treated with carbohydrate-based medicinal products worldwide. Direct regioselective acylation of methyl α-D-mannopyranoside (MDMP) derivatives 2-6 afforded from the 6-O-butyryl derivative. This isolated 6-O-derivative was converted to 2,3,4-tri-O-acyl derivatives, and the resulting compounds were analyzed using FTIR, 1H-NMR, 13C-NMR, and elemental analysis. The acylated derivatives showed moderate to good antimicrobial activity. Cytotoxicity assessment indicated that compound 2 had the lowest toxicity. A SAR study demonstrated that lauroyl and myristoyl acyl chains combined with mannopyranose were particularly effective against bacteria. In this context, molecular docking analysis demonstrated crucial interactions involved in assessing the binding affinity of ligands 1-6 for the active sites of Escherichia coli (4XO8) and Aspergillus flavus (1R51). A 100-ns molecular dynamics simulation showed that all the compounds were stable at the active site of protein 1R51. In silico ADMET prediction revealed greater drug similarity for MDMP derivatives. The results of this investigation may help create MDMP derivative-based multidrug-resistant antimicrobial agents.
... Further stabilization ensued via van der Waals interactions (His120, His23, Tyr80, and Asn25) and hydrophobic forces (Trp41, Phe172, and Tyr151). LasA is known for its proteolytic and elastolytic functions [50]. Operating as staphylolytic endopeptidase, LasA is known to cleave the pentaglycine bridge of peptidoglycan, alongside exhibiting elastinolytic activity [51]. ...
Antimicrobial resistance poses a severe threat, particularly in developing countries where the ready availability of drugs and increased consumption lead to improper antibiotic usage, thereby causing a surge in resistance levels compared to developed areas. Despite the past success of antibiotics, their effectiveness diminishes with regular use, posing a significant threat to medical efficacy. Pseudomonas aeruginosa, an opportunistic pathogen, triggers various infection-related issues, occurring on occasions including chronic wounds, burn injuries, respiratory problems in cystic fibrosis, and corneal infections. Targeting the quorum sensing (QS) of P. aeruginosa emerges as a strategic approach to combat infections caused by this bacterium. The objective of this study was to check the effect of antimycobacterial drugs against the potential QS targets in P. aeruginosa and identify lead candidates. The antimycobacterial drugs were first examined for the toxicological and pharmacokinetic profile. By virtual screening through molecular docking, delamanid and pretomanid stood out as major candidates. The binding energies of delamanid and pretomanid with LasR were determined to be −8.3 and −10.9 kcal/mol, respectively. The detailed analysis of the complexes of lead compounds were examined through molecular dynamics simulations. The molecular simulations data validated a sustained interaction of lead drugs with target proteins (PqsR, LasI, and LasA) in a physiological environment. The negligible changes in the secondary structure of proteins in presence of hit antimycobacterial drugs further strengthened the stability of the complexes. These findings highlight the potential repurposing of delamanid and pretomanid, specifically in targeting P. aeruginosa quorum-sensing mechanisms.
... Evaluation of the relative levels of pyoverdin in P. aeruginosa PAO1 followed a previously established protocol. 28 In summary, an overnight cultivated culture of P. aeruginosa PAO1, grown both in the absence and presence of 8-64 mg ml −1 TiO 2 -NPs, was collected. The culture was subjected to centrifugation to obtain the cell-free supernatant. ...
Antimicrobial resistance (AMR), a condition in which the efficacy of antimicrobial drugs in fighting microorganisms is reduced, has become a global challenge. Multidrug resistance (MDR) has been developing in microorganisms, where they can resist multiple medications. In particular, there has been a rise in MDR as well as extensively drug-resistant (XDR) strains of Pseudomonas aeruginosa in some regions, with prevalence rates ranging from 15% to 30%. The application of nanotechnology ranges from diagnostics to drug-delivery systems, revolutionizing healthcare, and improving disease treatment. We aimed to investigate the efficacy of titanium dioxide nanoparticles (TiO2-NPs) against various virulent traits of P. aeruginosa and S. marcescens. More than 50% reduction in the production of virulent pigments of P. aeruginosa was recorded following the treatment of TiO2-NPs. Additionally, elastases and exoproteases were inhibited by 58.21 and 74.36%, respectively. A similar result was observed against the rhamnolipid production and swimming motility of P. aeruginosa. The effect of TiO2-NPs was also validated against another opportunistic pathogen, S. marcescens, where the production of prodigiosin was reduced by 64.78%. Also, a roughly 75% attenuation of proteolytic activity and more than 50% reduction in swarming motility were found. In the control group, the cell surface hydrophobicity was 77.72%, which decreased to 24.67% with the addition of 64 μg ml⁻¹ TiO2-NPs in culture media. The hydrophobicity index of microorganisms is crucial for their initial attachment and the formation of biofilms. In conclusion, TiO2-NPs demonstrated potential in a multi-target approach against P. aeruginosa and S. marcescens, suggesting their advantages in the prevention and treatment of infections. These nanomaterials could have vital importance in the development of novel antibacterial agents to combat drug-resistant bacteria.
... The assessment of pyoverdin production followed a previously described method [26]. In brief, an overnight-cultured sample of P. aeruginosa PAO1, cultured in the absence and presence of Ti-Ce-NCs as mentioned earlier, was collected. ...
Antimicrobial resistance (AMR) has become a critical global health challenge. Infections, particularly those caused by multidrug-resistant (MDR) pathogens, rank among the top causes of human mortality worldwide. Pseudomonas aeruginosa occupies a prominent position among pathogens responsible for opportunistic infections in humans. P. aeruginosa stands as a primary cause of chronic respiratory infections, significantly contributing to the burden of these chronic diseases. In the medical domain, nanotechnologies offer significant potential, spanning various applications, including advanced imaging, diagnostic devices, drug delivery systems, implants, tissue-engineered structures, and pharmaceutical treatments. Given the challenges associated with AMR and the limited discovery of new drugs to combat MDR microbes, there is a critical need for alternative strategies to address the problem of AMR. In this study, we synthesized titanium–cerium nanocomposites (Ti–Ce–NCs) using an eco-friendly green synthesis approach. X-ray diffraction (XRD) analysis confirmed the crystalline nature of the Ti–Ce–NCs and determined the particle size to be 17.07 nm. Electron microscopy revealed the size range of the particles to be 13 to 54 nm, where the majority of the particles were in the 20 to 25 nm range. Upon examining the composition, the Ti–Ce–NCs were determined to be composed of cerium, oxygen, and titanium, whose relative abundance were 36.86, 36.6, and 24.77% by weight, respectively. These nanocomposites were then evaluated for their effectiveness against various virulent traits and biofilms in P. aeruginosa. Out of six tested virulence factors, more than 50% inhibition of five virulence factors of P. aeruginosa was found. Roughly 60% inhibition of biofilm was also found in the presence of 400 µg/mL Ti–Ce–NCs. The nanocomposites also altered the biofilm architecture of the test bacterium. The success of this research opens doors for the potential use of such nanomaterials in the discovery of new antibacterial agents to combat drug-resistant bacteria.
... Biofilms are mainly composed of mucus secreted by the bacteria that envelopes them. With high resistance and low permeability to typical antimicrobial drugs, biofilms not only protect the bacteria from the host immune cells but also reduce the efficiency of antibiotic-based treatment, which in turn increases the risk of antibiotic resistance development [3,4]. The problem of the "failure" of antibiotics against biofilms and continuous enhancement of bacterial resistance are becoming increasingly prominent. ...
Background
Multidrug-resistant bacteria continue to emerge owing to the abuse of antibiotics and have a considerable negative impact on people and the environment. Bacteria can easily form biofilms to improve their survival, which reduces the efficacy of antibacterial drugs. Proteins such as endolysins and holins have been shown to have good antibacterial activity and effectively removal bacterial biofilms and reduce the production of drug-resistant bacteria. Recently, phages and their encoded lytic proteins have attracted attention as potential alternative antimicrobial agents. The aim of the present study was to investigate the sterilising efficacy of phages (SSE1, SGF2, and SGF3) and their encoded lytic proteins (lysozyme and holin), and to further explore their potential in combination with antibiotics. To the ultimate aim is to reduce or replace the use of antibiotics and provide more materials and options for sterilisation.
Results
Phages and their encoded lytic proteins were confirmed to have great advantages in sterilisation, and all exhibited significant potential for reducing bacterial resistance. Previous studies on the host spectrum demonstrated the bactericidal efficacy of three Shigella phages (SSE1, SGF2, and SGF3) and two lytic proteins (LysSSE1 and HolSSE1). In this study, we investigated the bactericidal effects on planktonic bacteria and bacterial biofilms. A combined sterilisation application of antibiotics, phages, and lytic proteins was performed. The results showed that phages and lytic proteins had better sterilisation effects than antibiotics with 1/2 minimum inhibitory concentrations (MIC) and their effect was further improved when used together with antibiotics. The best synergy was shown when combined with β- lactam antibiotics, which might be related to their mechanism of sterilising action. This approach ensures a bactericidal effect at low antibiotic concentrations.
Conclusions
This study strengthens the idea that phages and lytic proteins can significantly sterilise bacteria in vitro and achieve synergistic sterilisation effects with specific antibiotics. Therefore, a suitable combination strategy may decrease the risk of drug resistance.
... The radius of gyration is the mass-weighted RMS distance of the collection of atoms from their common center of mass. [46] R g is one of the important parameters to analyze the stability of proteins using MD simulation data. [47] The R g of α-demethylase and Uox alone and all their complexes are shown in Fig. 10a. ...
Nucleoside analogs are an important, well-established class of clinically useful medicinal agents that exhibit potent antimicrobial activity. Thus, we designed to explore the synthesis and spectral characterization of 5'-O-(myristoyl)thymidine esters (2-6) for in vitro antimicrobial, molecular docking, molecular dynamics, SAR, and POM analyses. An unimolar myristoylation of thymidine under controlled conditions furnished the 5'-O-(myristoyl)thymidine and it was further converted into four 3'-O-(acyl)-5'-O-(myristoyl)thymidine analogs. The chemical structures of the synthesized analogs were ascertained by analyzing their physicochemical, elemental, and spectroscopic data. In vitro antimicrobial tests along with PASS, prediction indicated expectant antibacterial functionality of these thymidine esters compared to the antifungal activities. In support of this observation, their molecular docking studies have been performed against lanosterol 14α-demethylase (CYP51A1) and Aspergillus flavus (1R51) and significant binding affinities and non-bonding interactions were observed. The stability of the protein-ligand complexes was monitored by a 100 ns MD simulation and found the stable conformation and binding mode in a stimulating environment of thymidine esters. Pharmacokinetic predictions were studied to assess their ADMET properties and showed promising results in silico. SAR investigation indicated that acyl chains, lauroyl (C-12) and myristoyl (C-14), combined with deoxyribose, were most effective against the tested bacterial and fungal pathogens. The POM analyses provide the structural features responsible for their combined antibacterial/antifungal activity and provide guidelines for further modifications, with the aim of improving each activity and selectivity of designed drugs targeting potentially drug-resistant microorganisms. It also opens avenues for the development of newer antimicrobial agents targeting bacterial and fungal pathogens.
... The radius of gyration (R g ) is described mass-weighted root mean square distance of collection of atoms from their common centre of mass. R g is an important indicator in examining the stability of proteins [48]. R g of α-cry alone and α-cry-butein complex is shown in Fig. 4D. ...
The aggregation of lens proteins induced by glycation is one of the key drivers of diabetic retinopathy and development of diabetic cataracts. Moreover, glycation also causes numerous alterations not only to the tertiary structure of lens proteins but also to serum proteins. There are also evidences of covalent crosslinking among lens crystallins resulting in development of cataract. In this article, the inhibitory potential of butein was tested against the glucose induced glycation and the aggregation α-crystallin (α-cry). The results showed that there was inhibition of advanced glycation products (78.28%) and early glycation products (86.30%) following the treatment of butein. Additionally, the presence of butein caused a significant improvement in the tested biochemical markers of glycation. The treatment with butein reduced the free lysine modification to 23.67%. The secondary and tertiary structural distortions of α-cry were also protected. The mechanism of inhibition further investigated at the molecular level using biophysical and computational techniques. The interaction data showed the butein exhibited strong affinity towards the α-cry. The binding event was entropically driven and energetically favourable. The Gibb’s free energy of the interaction was found to be -5.99 to -7.17 kcal mol⁻¹. The binding site of butein in α-cry was deciphered by molecular docking and the dynamics was studied using molecular dynamics (MD) simulations. The simulation data showed that butein formed stable complex with α-cry under physiological conditions. Most of the tested parameters from molecular simulations, such as secondary structure, was found to be stable. The data clearly show the potential of butein in inhibiting the glycation induced aggregation of α-cry and hence can be developed as useful inhibitor in the management of diabetic cataract and retinopathy.
... Recently, one MTb natural metabolite Pht analog Plumbagin secreted by Plumbago zeylanica L. was identified to inhibit P. aeruginosa LasR protein-regulated las QS system (Qais et al., 2021). In this study, we reported the dual-functional P. aeruginosa PQS and MTb Pht interact with host AhR and bacterial PqsR proteins (Figure 8). ...
The aryl hydrocarbon receptor (AhR) protein senses microbial-secreted metabolites to trigger the host's innate immune system. The Pseudomonas quinolone signal (PQS) and Mycobacterium tuberculosis (MTb) metabolite phthiocol (Pht) are both ligands of AhR with similar chemical structures. As PQS is an essential quorum-sensing molecule that regulates a wide range of virulence factors in Pseudomonas aeruginosa, we hypothesized that Pht and its analogs are potential P. aeruginosa quorum-sensing inhibitors (QSIs) with immune-modulating functions. In this study, we demonstrated that Pht was able to inhibit the P. aeruginosa pqs QS system and reduce both biofilm formation and the production of pyocyanin. Molecular docking analysis suggested that Pht competes with PQS at the binding site of its receptor, PqsR. An electrophoretic mobility shift assay confirmed the Pht-PqsR interaction and showed that Pht attenuated PqsR from binding to the pqsA promoter. Proteomic analysis showed that synthesis of the key pqs QS proteins decreased upon the addition of Pht to the bacterial cultures. Furthermore, Pht analogs vitamins K1 (Phylloquinone), K2 (Menaquinones), and K3 (Menadione) were also showed to inhibit the P. aeruginosa pqs QS system while able to activate the AhR signaling pathways. Our study suggests that the AhR ligands Pht and its vitamin K analogs are promising QSIs for the alternative treatment of P. aeruginosa infections.
... R g is defined as the mass-weighted RMS distance of the collection of atoms from their common centre of mass. 62 R g is an important parameter in studying the stability of proteins and complexes in MD simulation studies. 63 The R g of BuChE and its complexes is presented in Figure 6a. ...
Currently, anti-butyrylcholinesterase is among the greatest therapeutic agents for the treatment of Alzheimer’s disease. In this research, a series of thirty-six carbamate derivatives were subjected to a QSAR study using DFT and Lipinski’s descriptors. Multiple linear regression was used to explore the relationships between the structural features of these compounds and butylcholinesterase inhibitory activity. In order to generate results applicable in the experimental plan, the OECD guidelines were adopted. The quality of MLR model was evaluated by several statistical parameters including internal and external validation parameters (R2, R2adj, F, VIF, R2test, Q2CV, R2Rand, Q2CV (Rand) and cRp2). The built model displayed a high predictive power (R2test = 0.817). What is more, the internal validation parameters (Q2CV = 0.774; average R2Rand = 0.118; average Q2CV (Rand) = -0.438; cRp2 =0.820) highlight the robustness of our built model. Besides, the obtained result revealed that Anti-butylcholinesterase activity is mainly attributed to the following molecular destructors: Octanol-Water partition (LogP), highest occupied molecular orbital energy (ELOMO), total energy (ET) and dipole moment (µ). Based on these findings, a series of newer synthesizable compounds with enhanced Anti-BuChE activities were designed and their ADMET and drug-likeness properties were further predicted to filter out compounds likely to fail during drug development stages. Finally, a molecular docking was performed to identify the binding types between the best designed compounds and BuChE enzyme.
The emergence of multidrug resistance (MDR) in bacterial pathogens is a serious public health concern. A significant therapeutic target for MDR infections is the quorum sensing‐regulated bacterial pathogenicity. Determining the anti‐quorum sensing abilities of certain medicinal plants against bacterial pathogens as well as the in‐silico interactions of particular bioactive phytocompounds with QS and biofilm‐associated proteins were the objectives of the present study. In this study, 6 medicinal plants were selected based on their ethnopharmacological usage, screened for Anti‐QS activity and Artemisia annua leaf extract (AALE) demonstrated pigment inhibitory activity against Chromobacterium violaceum CV12472. Further, the methanol active fraction significantly inhibited the virulence factors (pyocyanin, pyoverdine, rhamnolipid and swarming motility) of Pseudomonas aeruginosa PAO1 and Serratia marcescens MTCC 97 at respective sub‐MICs. The inhibition of biofilm was determined using a microtiter plate test and scanning electron microscopy. Biofilm formation was impaired by 70%, 72% and 74% in P. aeruginosa , C. violaceum and S. marcescens , respectively at 0.5xMIC of the extract. The phytochemical content of the extract was studied using GC–MS and 1, 8‐cineole was identified as major bioactive compound. Furthermore, 1, 8‐cineole was docked with quorum sensing (QS) proteins (LasI, LasR, CviR, and rhlR) and biofilm proteins (PilY1 and PilT). In silico docking and dynamics simulations studies suggested interactions with QS‐receptors CviR’, LasI, LasR, and biofilm proteins PilY1, PilT for anti‐QS activity. Further, 1, 8‐cineole demonstrated 66% and 51% reduction in violacein production and biofilm formation, respectively to validate the findings of computational analysis. Findings of the present investigation suggests that 1, 8‐cineole plays a crucial role in the QS and biofilm inhibitory activity demonstrated by Artemisia annua extract.
Research Highlights
Artemisia annua leaf extract (AALE) methanol fraction demonstrated broad‐spectrum QS and biofilm inhibition
Scanning electron microscopy (SEM) confirmed biofilm inhibition
Molecular docking and simulation studies suggested positive interactions of 1,8‐cineol with QS‐receptors and biofilm proteins
In last two decades, the world has seen an exponential increase in the antimicrobial resistance (AMR), making the issue a serious threat to human health. The mortality caused by AMR is one of the leading causes of human death worldwide. Till the end of the twentieth century, a tremendous success in the discovery of new antibiotics was seen, but in last two decades, there is negligible progress in this direction. The increase in AMR combined with slow progress of antibiotic drug discovery has created an urgent demand to search for newer methods of intervention to combat infectious diseases. One of such approach is to look for biofilm and quorum sensing (QS) inhibitors. Plants are excellent source of wide class compounds that can be harnessed to look for the compounds with such properties. This study proves a broad-spectrum biofilm and QS inhibitory potential of umbelliferone. More than 85% reduction in violacein production Chromobacterium violaceum 12472 was found. All tested virulent traits of Pseudomonas aeruginosa PAO1 and Serratia marcescens MTCC 97 were remarkably inhibited that ranged from 56.62% to 86.24%. Umbelliferone also successfully prevented the biofilm of test bacteria at least by 67.68%. Umbelliferone interacted at the active site of many proteins of QS circuit, which led to the mitigation of virulent traits. The stable nature of complexes of umbelliferone with proteins further strengthens in vitro results. After examining the toxicological profile and other drug-like properties, umbelliferone could be potentially developed as new drug to target the infections caused by Gram - ve bacteria.Communicated by Ramaswamy H. Sarma.
The environmental biotic and abiotic factors form a complicated relationship with the host intestinal microbiota. In our study, we applied different levels of C/N ratio (10, 15, 20) and frequent addition times (once, twice, triple a day) in a factorial experimental design. GC/LC analysis of filtrated biofloc (BF) samples revealed the highest relative fold change for the untargeted bioactive molecules among different treatments, whereas the 16s rRNA analysis revealed the change in the shrimp gut microbiota composition. Based on the available literature on the relationship between the bioactive molecules and the available bacteria in this study, the next bioactive molecules were discussed. Proline was associated with Bacteroidota, Flavobacteriaceae, Gammaproteobacteria, and Flavobacteriales. Plumbagine was associated with Norcardiaceae. Phytosphingosin was associated with Bacteroidota. Phosphocholine compound was associated with Bacteroidota. The monobutyl ether, benzofuran, and piperidone were associated with Micobacteriaceae and Mycobacterium. Generally, C/N 15 and 20 once a day, and C/N 20 triple a day have showed a merit over other treatments in term of low pathogenic and unfavorable bacteria, and high commensal bacterial abundances. The revealed bioactive molecule composition showed the complicity of BF as a source for novel compounds as biosecurity agents in BF system. These molecules could be developed to feed additives upgrading the biosecurity level in aquaculture systems. Other bioactive molecules require future studies to reveal novel molecules in term of aquaculture biosecurity control.
Antimicrobial resistance threatens the effective prevention and treatment of an ever‐increasing range of infections caused by different types of bacteria. Addressing antibacterial resistance and protecting human health requires multi‐sectoral, long‐acting, and professional administration. Sustainable investment in the R&D of antimicrobial drugs is one of the critical strategies for solving the current problem. Specifically, the development of novel effective drug candidates with a unique mode of action is essential for overcoming antibacterial resistance. 1,4‐Naphthoquinone is a quinone derivative from naphthalene, one of the most important subclasses of quinone. 1,4‐Naphthoquinones have been studied as potential candidates against various diseases, including cancer and malaria, and are a promising new compound that can treat bacterial infections. This review focuses on 1,4‐naphthoquinone analogs for their activity against bacteria.
The glycation and aggregation of lens proteins significantly contribute to the onset of diabetic cataracts as well as the retinopathy. The glycation exerts numerous alterations in the tertiary structural of proteins. Moreover, the covalent crosslinking of lens crystallins also contribute to the cataract formation. In this article, the effect of pioglitazone on glucose induced glycation and aggregation α-crystallin was examined. A remarkable inhibition of early glycation products (~80%) and advanced glycation products (~75%) was recorded by the treatment of pioglitazone. There was >75% recovery in biochemical marker (carbonyl content). The presence of 150 μM of pioglitazone reduced the free lysine modifications to 35%. Treatment of pioglitazone also protected the secondary structural alterations induced by glycation and inhibited the formation of protein aggregates. The interaction studies showed that pioglitazone interacted with α-crystallin via moderate binding affinity. The interaction between pioglitazone interacted and α-crystallin was energetically and entropically favourable. The complex of pioglitazone with studied protein stable in which RMSF, Rg, SASA, RMSD, and the secondary structural components was not affected. The findings show antiglycation activity of pioglitazone along with its mechanism of action highlighting the ability of drug to be possibly developed novel as glycation inhibitor.
Monophasic tin dioxide nanoflowers (TONFs) assembled by rod-like nanostructures were prepared by coprecipitation method using tin chloride and ammonia precipitators, as the starting materials, without using any surfactants or templates. The structural, compositional, optical, and morphological properties of TONFs were investigated by XRD, FT-IR, UV-vis, SEM-EDX, and TEM techniques. Synthesized TONFs demonstrated inhibition of quorum sensing- (QS-) regulated virulence in pathogens, viz., Chromobacterium violaceum , Pseudomonas aeruginosa , and Serratia marcescens . Significant reduction in biofilm formation in all test pathogens was also observed which was further validated by CLSM images illustrating disturbed biofilm architecture. Vital functions like EPS, swarming motility, and cell surface hydrophobicity that contribute to successful biofilm formation were reduced after addition of sub-MICs of TONFs. Significant eradication of preformed biofilms was recorded upon addition of subinhibitory concentrations of TONFs in all test pathogens. The study highlights the broad-spectrum QS and biofilm inhibition by TONFs that can be exploited in future to protect food from contamination and combatting persistent drug-resistant infections.
Pseudomonas aeruginosa (P.a) is a pathogen causing significant morbidity and mortality, in particular, in hospital patients undergoing ventilation and in patients with cystic fibrosis. Among the virulence factors secreted or injected into host cells, the physiopathological relevance of type II secretions system (T2SS) is less studied. Although there is extensive literature on the destructive role of LasB in vitro on secreted innate immune components and on some stromal cell receptors, studies on its direct action on myeloid cells are scant. Using a variety of methods, including the use of bacterial mutants, gene-targeted mice, and proteomics technology, we show here, using non-opsonic conditions (thus mimicking resting and naïve conditions in the alveolar space), that LasB, an important component of the P.a T2SS is highly virulent in vivo, and can subvert alveolar macrophage (AM) activity and bacterial killing, in vitro and in vivo by downregulating important secreted innate immune molecules (complement factors, cytokines, etc.) and receptors (IFNAR, Csf1r, etc.). In particular, we show that LasB downregulates the production of C3 and factor B complement molecules, as well as the activation of reactive oxygen species production by AM. In addition, we showed that purified LasB impaired significantly the ability of AM to clear an unrelated bacterium, namely Streptococcus pneumoniae. These data provide a new mechanism of action for LasB, potentially partly explaining the early onset of P.a, alone, or with other bacteria, within the alveolar lumen in susceptible individuals, such as ventilated, chronic obstructive pulmonary disease and cystic fibrosis patients.
Background
Pseudomonas aeruginosa lung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity.
Objective
We evaluated the effect of P. aeruginosa elastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo.
Methods
Wild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or ΔLasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models.
Results
We showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways.
Conclusions
Our data demonstrate that PAO1 LasB is a major P. aeruginosa secreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals.
Quorum sensing (QS) is a global gene regulatory mechanism in bacteria for various traits including virulence factors. Disabling QS system with anti-infective agent is considered as a potential strategy to prevent bacterial infection. Mangifera indica L. (mango) has been shown to possess various biological activities including anti-QS. This study investigates the efficacy of leaf extracts on QS-regulated virulence factors and biofilm formation in Gram negative pathogens. Mango leaf (ML) extract was tested for QS inhibition and QS-regulated virulence factors using various indicator strains. It was further correlated with the biofilm inhibition and confirmed by electron microscopy. Phytochemical analysis was carried out using ultra performance liquid chromatography (UPLC) and gas chromatography–mass spectrometry (GC-MS) analysis. In vitro evaluation of anti-QS activity of ML extracts against Chromobacterium violaceum revealed promising dose-dependent interference in violacein production, by methanol extract. QS inhibitory activity is also demonstrated by reduction in elastase (76%), total protease (56%), pyocyanin (89%), chitinase (55%), exopolysaccharide production (58%) and swarming motility (74%) in Pseudomonas aeruginosa PAO1 at 800 μg/ml concentration. Biofilm formation by P. aeruginosa PAO1 and Aeromonas hydrophila WAF38 was reduced considerably (36–82%) over control. The inhibition of biofilm was also observed by scanning electron microscopy. Moreover, ML extracts significantly reduced mortality of Caenorhabditis elegans pre-infected with PAO1 at the tested concentration. Phytochemical analysis of active extracts revealed very high content of phenolics in methanol extract and a total of 14 compounds were detected by GC-MS and UPLC. These findings suggest that phytochemicals from the ML could provide bioactive anti-infective and needs further investigation to isolate and uncover their therapeutic efficacy.
Aim:
Pseudomonas aeruginosais one of the fatal biofilm forming pathogens which poses to be a problem in clinical infections, contamination of food and marine ecosystems. In this report a napthoquinone – Plumbagin has been explored for its antimicrobial (antibacterial and antibiofilm) activity against P. aeruginosa biofilm. The ability of Plumbagin to enhance the bioactivity of a known broad spectrum antibiotic was further assayed by combining the sub-MIC doses of Plumbagin with sub-MIC doses of Gentamicin against P. aeruginosa biofilm.
Methods and Results:
This combinatorial approach was used for a series of experiments for understanding the mechanism of action for antibiofilm activity against P. aeruginosa(MTCC424, MTCC2488). Antibiofilm activity was studied by safranin staining, estimating total protein, visualisation of biofilms and EPS quantification. Antivirulent activity of these doses was studied by azocasein degradation, expression of virulent factors and molecular docking. Expression of quorum sensing (QS) phenotypes was studied by motility assessment and mRNA expression pattern of virulence genes. It was observed that Plumbagin alone and the combinatorial doses of Plumbagin and Gentamicin exhibit significant antibiofilm and antivirulent activity coupled with the reduction in expression of QS phenotypes and virulence genes. Molecular docking study revealed that Plumbagin had variable affinityfor different QS proteins.
Conclusion:
Low doses of Plumbagin and Gentamicin exhibit synergistic activity against P. aeruginosa biofilm whilst maintaining their effectiveness.
Significance and Impact of the study:
As the Pseudomonas aeruginosabiofilms are reservoir of persister bacteria, thus, the increasing concern of antibiotic tolerance has to be dealt with combinatorial approaches. In this report, Plumbagin has been explored in potentiating the antibiofilm effect of a broad spectrum antibiotic gentamicin for better therapeutic efficacy.
Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a plant-derived naphthoquinones obtained mainly from three families, including Plumbaginaceae, Droseraceae, and Ebenaceae. Plumbagin has exhibited its potential therapeutic benefits on numerous chronic diseases, i.e., breast cancer, non-small cell lung cancer, melanoma, ovarian, squamous cell carcinomas, pancreatic cancer, and prostate cancer. In addition, its anti-inflammatory and antimicrobial activities as well as control of diabetes and cardiovascular diseases have been reported. Thus, plumbagin is a promising agent for development as a new drug for the treatment or control of chronic diseases. Studies on controlled drug release or drug delivery systems have been involved for improvement of its therapeutic efficacy as well as for the reduction of its toxicity. However, most of the recent research information is from in vitro and in vivo studies. Further clinical studies are therefore required for its developments and applications as a novel drug used to treat chronic diseases.
Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to biocides used as disinfectants, antiseptics and preservatives, and to heavy metals (particularly copper and zinc) used as growth promoters and therapeutic agents for some livestock species. There is indeed experimental and observational evidence that exposure to these non-antibiotic antimicrobial agents can induce or select for bacterial adaptations that result in decreased susceptibility to one or more antibiotics. This may occur via cellular mechanisms that are protective across multiple classes of antimicrobial agents or by selection of genetic determinants for resistance to non-antibiotic agents that are linked to genes for antibiotic resistance. There may also be relevant effects of these antimicrobial agents on bacterial community structure and via non-specific mechanisms such as mobilization of genetic elements or mutagenesis. Notably, some co-selective adaptations have adverse
The present study was undertaken in order to understand more about the interaction occurring between S. maltophilia and P. aeruginosa, which are frequently co-isolated from CF airways. For this purpose, S. maltophilia RR7 and P. aeruginosa RR8 strains, co-isolated from the lung of a chronically infected CF patient during a pulmonary exacerbation episode, were evaluated for reciprocal effect during planktonic growth, adhesion and biofilm formation onto both polystyrene and CF bronchial cell monolayer, motility, as well as for gene expression in mixed biofilms. P. aeruginosa significantly affected S. maltophilia growth in both planktonic and biofilm cultures, due to an inhibitory activity probably requiring direct contact. Conversely, no effect was observed on P. aeruginosa by S. maltophilia. Compared with monocultures, the adhesiveness of P. aeruginosa on CFBE41o- cells was significantly reduced by S. maltophilia, which probably acts by reducing P. aeruginosa's swimming motility. An opposite trend was observed for biofilm formation, confirming the findings obtained using polystyrene. When grown in mixed biofilm with S. maltophilia, P. aeruginosa significantly over-expressed aprA, and algD-codifying for protease and alginate, respectively-while the quorum sensing related rhlR and lasI genes were down-regulated. The induced alginate expression by P. aeruginosa might be responsible for the protection of S. maltophilia against tobramycin activity we observed in mixed biofilms. Taken together, our results suggest that the existence of reciprocal interference of S. maltophilia and P. aeruginosa in CF lung is plausible. In particular, S. maltophilia might confer some selective "fitness advantage" to P. aeruginosa under the specific conditions of chronic infection or, alternatively, increase the virulence of P. aeruginosa thus leading to pulmonary exacerbation.
To explore the inhibitory mechanism of catechins for digestive enzymes, we investigated the binding mode of catechins to a typical digestive enzyme-trypsin and analyzed the structure-activity relationship of catechins, using an integration of molecular docking, molecular dynamics simulation and binding free energy calculation. We found that catechins with different structures bound to a conservative pocket S1 of trypsin, which is comprised of residues 189-195, 214-220 and 225-228. In the trypsin-catechin complexes, Asp189 by forming strong hydrogen bonding, and Gln192, Trp215 and Gly216 through hydrophobic interactions, all significantly contribute to the binding of catechins. The number and the position of hydroxyl and aromatic groups, the structure of stereoisomers, and the orientation of catechins in the binding pocket S1 of trypsin all affect the binding affinity. The binding affinity is in the order of Epigallocatechin gallate (EGCG) > Epicatechin gallate (ECG) > Epicatechin (EC) > Epigallocatechin (EGC), and 2R-3R EGCG shows the strongest binding affinity out of other stereoisomers. Meanwhile, the synergic conformational changes of residues and catechins were also analyzed. These findings will be helpful in understanding the knowledge of interactions between catechins and trypsin and referable for the design of novel polyphenol based functional food and nutriceutical formulas.
Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation.
Pseudomonas aeruginosa causes severe and persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection is hard to eradicate as P. aeruginosa has developed strong resistance to most conventional antibiotics. The problem is further compounded by the ability of the pathogen to form biofilm matrix, which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing (QS), a cell density-based intercellular communication system, which plays a key role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infection. The QS network of P. aeruginosa is organized in a multi-layered hierarchy consisting of at least four interconnected signaling mechanisms. Evidence is accumulating that the QS regulatory network not only responds to bacterial population changes but also could react to environmental stress cues. This plasticity should be taken into consideration during exploration and development of anti-QS therapeutics.
In the present study, the origin of the anti-quorum sensing (QS) activities
of several members of a recently synthesized and in vitro tested class of
lactone and thiolactone based inhibitors were computationally investigated.
Docking and molecular dynamic (MD) simulations and binding free energy
calculations were carried out to reveal the exact binding and inhibitory
profiles of these compounds. The higher in vitro activity of the lactone series
relative to their thiolactone isosteres was verified based on estimating the
binding energies, the docking scores and monitoring the stability of the
complexes produced in the MD simulations. The strong electrostatic contribution
to the binding energies may be responsible for the higher inhibitory activity
of the lactone with respect to the thiolactone series. The results of this
study help to understand the anti-QS properties of lactone-based inhibitors and
provide important information that may assist in the synthesis of novel QS
inhibitors.
Pseudomonas aeruginosa is the most common pathogen that persists in the cystic fibrosis lungs. Bacteria such as P. aeruginosa secrete siderophores (iron-chelating molecules) and the host limits bacterial growth by producing neutrophil-gelatinase-associated lipocalin (NGAL) that specifically scavenges bacterial siderophores, therefore preventing bacteria from establishing infection. P. aeruginosa produces a major siderophore known as pyoverdine, found to be important for bacterial virulence and biofilm development. We report that pyoverdine did not bind to NGAL, as measured by tryptophan fluorescence quenching, while enterobactin bound to NGAL effectively causing a strong response. The experimental data indicate that pyoverdine evades NGAL recognition. We then employed a molecular modeling approach to simulate the binding of pyoverdine to human NGAL using NGAL's published crystal structures. The docking of pyoverdine to NGAL predicted nine different docking positions; however, neither apo- nor ferric forms of pyoverdine docked into the ligand-binding site in the calyx of NGAL where siderophores are known to bind. The molecular modeling results offer structural support that pyoverdine does not bind to NGAL, confirming the results obtained in the tryptophan quenching assay. The data suggest that pyoverdine is a stealth siderophore that evades NGAL recognition allowing P. aeruginosa to establish chronic infections in CF lungs.
While much is known about how virulence factors affect pathogens and host tissues in vitro, far less is understood about their dynamics in vivo. As a step towards characterizing the chemistry of infected environments, we measured phenazine abundance in the lungs of cystic fibrosis patients. Phenazines are redox-active small molecules produced by Pseudomonas aeruginosa that damage host epithelia, curb the growth of competing organisms, and play physiologically important roles for the cells that produce them. Here, we quantify phenazines within expectorated sputum, characterize the P. aeruginosa populations responsible for phenazine production, and assess their broad effects on the CF lung microflora. Chemical analysis of expectorated sputum showed that the concentrations of two phenazines - pyocyanin (PYO) and phenazine-1-carboxylic acid (PCA) - negatively correlate (ρ = -0.68 and -0.57, respectively) with lung function. Furthermore, the highest phenazine concentrations were found in patients whose pulmonary function showed the greatest rate of decline. The constituent P. aeruginosa populations within each patient showed diverse phenazine production capacity: early in infection, individual isolates produced more PYO than later in infection, however, total PYO concentrations in sputum at any given stage correlated well with the average production by the total P. aeruginosa population. Finally, bacterial community complexity negatively correlated with phenazine concentrations and lung function decline, suggesting a link to the refinement of the overall microbial population. Together, these data demonstrate that phenazines negatively correlate with CF disease states in ways that were previously unknown, and underscore the importance of defining in vivo environmental parameters to better predict clinical outcomes of infections.
Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-A⁻/⁻ mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitro and in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin.
Discovery of quorum sensing (QS) system to coordinate virulence and biofilm formation in bacterial pathogens has triggered search for safe, stable and non-toxic anti-QS compounds from natural products. Ethanolic extracts of 24 Indian medicinal plants were tested by agar well and disc diffusion assay for anti-QS activity using Chromobacterium violaceum (CV12472 and CVO26) reporter strains. AHL from C. violaceum CV31532 was isolated and partially purified for its use in CVO26 based bioassay. Effect on swarming-motility of Pseudomonas aeruginosa (PAO1) was also recorded at sub-MIC concentrations of extracts. Of the 24 medicinal plants screened Hemidesmus indicus (L.) Schult (root), Holarrhena antidysenterica (Roth) A.DC. (bark), Mangifera indica L. (seed) Punica granatum L. (pericarp) and Psoralea corylifolia L. (seed) demonstrated varying level of inhibition of violacein production in the reporter strains. Moreover, a significant reduction in swarms was recorded over control. The inhibition of violacein production and swarming motility may be due to direct or indirect interference on QS by active constituents or the interactive effect of different phytocompounds present in the extracts. These plant extracts may be selected for activity guided fractionation to identify and characterize the active principle.
Iron is an essential element for life but also serves as an environmental signal for biofilm development in the opportunistic
human pathogen Pseudomonas aeruginosa. Under iron-limiting conditions, P. aeruginosa displays enhanced twitching motility and forms flat unstructured biofilms. In this study, we present evidence suggesting
that iron-regulated production of the biosurfactant rhamnolipid is important to facilitate the formation of flat unstructured
biofilms. We show that under iron limitation the timing of rhamnolipid expression is shifted to the initial stages of biofilm
formation (versus later in biofilm development under iron-replete conditions) and results in increased bacterial surface motility.
In support of this observation, an rhlAB mutant defective in biosurfactant production showed less surface motility under iron-restricted conditions and developed
structured biofilms similar to those developed by the wild type under iron-replete conditions. These results highlight the
importance of biosurfactant production in determining the mature structure of P. aeruginosa biofilms under iron-limiting conditions.
Quorum sensing is a cell-cell communication process in which bacteria use the production and detection of extracellular chemicals called autoinducers to monitor cell population density. Quorum sensing allows bacteria to synchronize the gene expression of the group, and thus act in unison. Here, we review the mechanisms involved in quorum sensing with a focus on the Vibrio harveyi and Vibrio cholerae quorum-sensing systems. We discuss the differences between these two quorum-sensing systems and the differences between them and other paradigmatic bacterial signal transduction systems. We argue that the Vibrio quorum-sensing systems are optimally designed to precisely translate extracellular autoinducer information into internal changes in gene expression. We describe how studies of the V. harveyi and V. cholerae quorum-sensing systems have revealed some of the fundamental mechanisms underpinning the evolution of collective behaviors.
Actively growing Escherichia coli cells exposed to plumbagin, a redox cycling quinone that increases the flux of O2- radicals in the cell, were mutagenized or killed by this treatment. The toxicity of plumbagin was not found to be mediated by membrane damage. Cells pretreated with plumbagin could partially reactivate lambda phage damaged by exposure to riboflavin plus light, a treatment that produces active oxygen species. The result suggested the induction of a DNA repair response. Lambda phage damaged by H2O2 treatment were not reactivated in plumbagin-pretreated cells, nor did H2O2-pretreated cells reactivate lambda damaged by treatment with riboflavin plus light. Plumbagin treatment did not induce lambda phage in a lysogen, nor did it cause an increase in beta-galactosidase production in a dinD::Mu d(lac Ap) promoter fusion strain. Cells pretreated with nonlethal doses of plumbagin showed enhanced survival upon exposure to high concentrations of plumbagin, but were unchanged in their susceptibility to far-UV irradiation. polA and recA mutants were not significantly more sensitive than wild type to killing by plumbagin. However, xth-1 mutants were partially resistant to plumbagin toxicity. It is proposed that E. coli has an inducible DNA repair response specific for the type of oxidative damage generated during incubation with plumbagin. Furthermore, this response appears to be qualitatively distinct from the SOS response and the repair response induced by H2O2.
Bacterial infections associated with indwelling medical devices often demonstrate an intrinsic resistance to antimicrobial
therapies. In order to explore the possibility of transport limitation to biofilm bacteria as a contributing factor, the penetration
of a fluoroquinolone antibiotic, ciprofloxacin, through Pseudomonas aeruginosa biofilms was investigated. Attenuated total
reflection Fourier transform infrared (ATR/FT-IR) spectrometry was employed to monitor bacterial colonization of a germanium
substratum, transport of ciprofloxacin to the biofilm-substratum interface, and interaction of biofilm components with the
antibiotic in a flowing system. Transport of the antibiotic to the biofilm-substratum interface during the 21-min exposure
to 100 micrograms/ml was found to be significantly impeded by the biofilm. Significant changes in IR bands of the biofilm
in regions of the spectrum associated with RNA and DNA vibrational modes appeared following exposure to the antibiotic, indicating
chemical modification of biofilm components. These results suggest that transport limitations may be an important factor in
the antimicrobial resistance of biofilm bacteria and that ATR/FT-IR spectrometry may be used to follow the time course of
antimicrobial action in biofilms in situ.
Studies that evaluated the mechanisms of action of Plumbagin (PLB) and its toxicity may contribute to future therapeutic applications of this compound. We investigate biomarker important in the mechanisms of action correlate the expression of mRNA with the cytotoxic and genotoxic effects of PLB on HepG2/C3A. In the analysis of cytotoxicity, PLB decreased cell viability and membrane integrity at concentrations ≥ 15μM. Xenobiotic-metabolizing system showed strong mRNA induction of CYP1A1, CYP1A2, and CYP3A4, suggesting extensive metabolization. PLB induced apoptosis and an increase in the mRNA expression of genes BBC3, CASP3, and CASP8. At a concentration of 15μM, there was a reduction in the expression of PARP1 mRNA and an increase in the expression of BECN1 mRNA, suggesting that PLB may also induce cell death by autophagy. PLB induced an arrest at the G2/M phase due to DNA damage, as observed in the comet assay. This damage is associated with the increased mRNA expression of genes p21, GADD45A, and H2AFX and with changes in the expression of proteins H2AX, p21, p53, Chk1, and Chk2. These results allow a better understanding of the cellular action of PLB and of its toxicity, thereby contributing to the development of PLB-based drugs, with markers of mRNA expression possibly playing a role as indicators for monitoring toxicity in human cells.
The biomolecular interaction of triamcinolone with bovine serum albumin (BSA) was performed using various multi-spectroscopic techniques in combination with in silico studies. UV-visible absorption, fluorescence spectroscopy and resonance Rayleigh scattering studies confirmed the formation of BSA- triamcinolone complex. The binding constant was found to be in the order of 103 M-1. Conformational and microenvironmental changes in BSA after addition of triamcinolone were confirmed by circular dichroism and 3D fluorescence spectroscopy, respectively. The negative values of ΔG and ΔH confirmed spontaneous and exothermic binding. The average binding distance between BSA and triamcinolone was also calculated through FRET. Additionally, the effect of metal ions and β-cyclodextrin on the binding of triamcinolone with BSA was also investigated. Molecular docking and site marker displacement experiments unveiled the binding of triamcinolone to BSA at site III located in subdomain IB of BSA. Molecular dynamics simulation showed lower RMSD value and negative total energy suggesting favourable binding between BSA and triamcinolone.
The emerging prevalence of multidrug-resistance in Gram-negative pathogens, due to conventional antimicrobial therapeutics, has led the researchers to emphasize on development of alternative novel strategies to suppress the bacterial virulence and pathogenicity through inhibition of quorum sensing (QS) and biofilms. QS is a bacterial communication system to produce density-dependent response via chemical signalling that controls pathogenesis and biofilms formation. Leaves of green tea are used worldwide as beverage which is also known for its broad-spectrum therapeutic efficacy. In this work, we have identified and characterized the most bioactive faction of green tea extract and evaluated the anti-QS and antibiofilm activity of green tea ethyl acetate fraction (GTEF) i.e. most active fraction, on three different Gram-negative bacterial pathogens. GTEF inhibited the violacein production by >75% in C. violaceum 12472. Many virulence factors of P. aeruginosa PAO1 viz. pyocyanin, pyoverdin, exoprotease, elastase, rhamnolipid production, and swimming motility were remarkably reduced in presence of sub-MICs of GTEF. Moreover, prodigiosin, protease activity, cell surface hydrophobicity, and swimming of S. marcescens MTCC 97 were also decreased significantly by the supplementation of GTEF in culture media. GTEF exhibited broad-spectrum antibiofilm action with >80% reduction in biofilm formation of test pathogens. In silico studies gave a mechanistic insight of action of GTEF. Molecular modelling revealed that phytoconstituents detected by GC/MS exhibited affinity (in order of 10⁴ M⁻¹) towards AHL synthases (LasI and EsaI). The molecular binding between phytocompounds and receptor proteins (LasR, RhlR, and PqsR) of QS circuit was also energetically favourable (ΔG°≥ 5.0 kcal mol⁻¹) and supported by hydrogen bonds and hydrophobic interactions. These compounds were found to be docked in ligand binding domain of CviR and occupied same cavity as that of its antagonist. Squalene and thunbergol interacted with LasA at tartaric acid binding pocket and the complex was strengthened with binding energy −5.9 kcal mol⁻¹. Moreover, interaction of thunbergol with biofilm-associated proteins viz. PilT and PilY1, might be disabling the pilus assembly and consequently inhibiting biofilm formation. In vivo validation of results suggested the protective role GTEF against QS-mediated pathogenicity and it might become a novel non-antibiotic QS inhibitor to control bacterial infection.
Cefotaxime is third generation antibiotic with known therapeutic efficacy against bacterial infections including cerebral abscesses and bacterial meningitis. The β-lactam group of drugs are considered safest antibiotics. Many antibiotics directly interact with DNA and alter their expression profile. Thus, it is necessary to understand the binding mode and its relevance to drug activity and toxicity. There is considerably a remarkable focus on deciphering the binding mechanism of these therapeutic agents as DNA is one of the major target for wide range of drugs. Cefotaxime has been extensively studied for its pharmacological properties while its binding mode to DNA has not been explicated so far. In this study, we have unveiled the binding mechanism of cefotaxime to DNA by using various biophysical, thermodynamic and in silico techniques. UV-vis spectroscopy confirmed the formation cefotaxime-DNA complex along with a brief idea about the extent of interaction. Fluorescence spectroscopy yielded the values of various binding constants and explained mode of fluorescence quenching to be static. CD spectroscopy, thermal denaturation, KI quenching and viscosity measurement explained that cefotaxime is groove binder. Measuring the effect of ions on cefotaxime-DNA complex ensured that it does not bind to DNA electrostatically. Dye displacement experiments finally confirmed that cefotaxime binds to the minor groove of DNA. ITC gave the thermodynamic profile of this binding in which negative value of Gibb's free energy change revealed that the process is spontaneous. Molecular modelling finally strengthened our experimental results that cefotaxime was located in curved contour of minor groove of DNA. The findings support on safety of drug and may have a little interference on normal biological functions.
Pseudomonas aeruginosa can cause complicated urinary tract infections, particularly in people with catheters, which can lead to pyelonephritis. Whilst some subgroups appear more susceptible to infection, such as the elderly and women, the contribution of other host factors and bacterial virulence factors to successful infection remains relatively understudied. In this review, we explore the potential role of P. aeruginosa virulence factors including phenazines, quorum sensing, biofilm formation, and siderophores along with host factors such as Tamm-Horsfall protein, osmotic stress and iron specifically on establishment of successful infection in the urinary niche. P. aeruginosa urinary tract infections are highly antibiotic resistant and require costly and intensive treatment. By understanding the infection dynamics of this organism within this specific niche, we may be able to identify novel therapeutic strategies to enhance the use of existing antibiotics.
Antibiotic resistance has become serious global threat to public health; therefore, the improved strategies and structurally novel antimicrobials are urgently needed to combat infectious diseases. Here we report a new type of highly potent 4-aminoquinoline derivatives as quorum sensing inhibitors in Serratia marcescens and Pseudomonas aeruginosa, exhibiting weak bactericidal activities (MIC > 400 ?M). Through detailed structure-activity study we have identified 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines (5 and 10) as biofilm formation inhibitors with 50% biofilm inhibition at 69 ?M and 63 ?M in S. marcescens and P. aeruginosa, respectively. These two compounds, 5 and 10, are the first quinoline derivatives with anti-biofilm formation activity reported in S. marcescens. QSAR analysis identified structural descriptors such as Wiener indices, HDPI, MTC, TCI, and logD(o/w)exp as the most influential in biofilm inhibition in this bacterial species. Derivative 10 is one of the most potent quinoline type inhibitors of pyocyanin production described so far (IC50 = 2.5 ?M). While we have demonstrated that 5 and 10 act as Pseudomonas quinolone system (PQS) antagonists, the mechanism of inhibition of S. marcescens biofilm formation with these compounds remains open since signaling similar to P. aeruginosa PQS system has not yet been described in Serratia and activity of these compounds on AHL signalling has not been detected. Our data show that 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines present the promising scaffolds for developing anti-virulence and anti-biofilm formation agents against multidrug-resistant bacterial species.
Studying the mode of interaction between small molecules and DNA have received much attention in recent years, as many drugs have been reported to directly interact with DNA thereby regulating the expression of many genes. Capsaicin is a capsaiciniods family phytocompound having many therapeutic applications including diabetic neuropathy, rheumatoid arthritis, prevention of DNA strand breaks and chromosomal aberrations. In this study, we have investigated the interaction of capsaicin with calf thymus DNA using a number of biophysical techniques to get an insight and better understanding of the interaction mechanism. Analysis of UV-visible absorbance spectra and fluorescence spectra indicates the formation of complex between capsaicin and Ct-DNA. Thermodynamic parameters ΔG, ΔH, and ΔS measurements were taken at different temperatures indicated that hydrogen bonding and van der Waal’s forces played major role in the binding process. Additional experiments such as iodide quenching, CD spectroscopy suggested that capsaicin possibly binds to the minor groove of the Ct-DNA. These observations were further confirmed by DNA melting studies, viscosity measurements. Molecular docking provided detailed computational interaction of capsaicin with Ct-DNA which proved that capsaicin binds to Ct-DNA at minor groove. Computational molecular docking also revealed the exact sites and groups to which capsaicin interacted.
Background:
Biofilms are ubiquitous, and have been observed in both acute and chronic wounds. Their role in wound healing and infection, however, remains controversial. The aim of this review was to provide an overview of the role and relevance of biofilms to surgical wounds.
Methods:
A search of PubMed, Science Direct and Web of Science databases was performed to identify studies related to biofilms. Specifically, studies were sought in acute and chronic wounds, and the management and treatment of non-healing and infected skin and wounds.
Results:
Biofilms may develop in all open wounds. In chronic wounds, biofilms may play a role in prolonging and preventing healing, causing chronic inflammation and increasing the risk of infection. Controversies exist regarding the methods presently employed for biofilm detection and management and few data exist to underpin these decisions.
Conclusion:
Biofilms in acute surgical and chronic wounds appear to cause a delay in healing and potentially increase the risk of infection. Biofilms can be prevented and once developed can be controlled using wound desloughing and debridement.
Ethnopharmacological relevance: Piper betle, a tropical creeper plant belongs to the family Piperaceae. The leaves of this plant have been well known for their therapeutic, religious and ceremonial value in South and Southeast Asia. It has also been reported to possess several biological activities including antimicrobial, antioxidant, antinociceptive, antidiabetic, insecticidal and gastroprotective activities and used as a common ingredient in indigenous medicines. In Indian system of ayurvedic medicine, P. betle has been well recognized for its antiseptic properties and is commonly applied on wounds and lesions for its healing effects.
Aim of the study: To evaluate the anti-quorum sensing (anti-QS) and antibiofilm efficacy of P. betle and its bioactive metabolite phytol against Serratia marcescens.
Materials and methods: The P. betle ethyl acetate extract (PBE) was evaluated for its anti-QS efficacy against S. marcescens by assessing the prodigiosin and lipase production at 400 and 500 µg ml-1 concentrations. In addition, the biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of PBE against S. marcescens. Besides, the influence of PBE on bacterial biofilm formation was assessed through microscopic techniques. The biofilm related phenomenons like exopolysaccharides (EPS) production, hydrophobicity and swarming motility were also examined to support the antibiofilm activity of PBE. Transcriptional analysis of QS regulated genes in S. marcescens was also done. Characterization of PBE was done by separation through column chromatography and identification of active metabolites by gas chromatography -mass spectrometry. The major compounds of active fractions such as hexadecanoic acid, eugenol and phytol were assessed for their anti-QS activity against S. marcescens. Further, the in vitro bioassays such as protease, biofilm and HI quantification were also carried out to confirm the anti-QS and antibiofilm potential of phytol in PBE.
Results: PBE inhibits QS mediated prodigiosin pigment production in S. marcescens, which confirmed its anti-QS potential against S. marcescens. At 500 µg ml-1 concentration, PBE significantly inhibited the production of protease, lipase, biofilm and EPS to the level of 71%, 68%, 65% and 43% in S. marcescens, respectively. Further, their antibiofilm efficacy was confirmed through microscopic techniques. In addition, PBE effectively inhibited the hydrophobicity and swarming motility. Additionally, the results of qPCR analysis validated the downregulation of QS genes. Chromatographic techniques the presence of hexadecanoic acid, eugenol and phytol in PBE and the potential bioactive compound with anti-QS activity was identified as phytol. In vitro assays with phytol evidenced the potent inhibition of QS-controlled prodigiosin, protease, biofilm and hydrophobicity in S. marcescens, without exerting any deleterious effect on its growth.
Conclusion: This study demonstrates the promising anti-QS and antibiofilm activities of PBE and its active metabolite phytol, and confirms the ethnopharmacological applications of these leaves against S. marcescens infections.
Bacteria use quorum sensing to orchestrate gene expression programmes that underlie collective behaviours. Quorum sensing relies on the production, release, detection and group-level response to extracellular signalling molecules, which are called autoinducers. Recent work has discovered new autoinducers in Gram-negative bacteria, shown how these molecules are recognized by cognate receptors, revealed new regulatory components that are embedded in canonical signalling circuits and identified novel regulatory network designs. In this Review we examine how, together, these features of quorum sensing signal-response systems combine to control collective behaviours in Gram-negative bacteria and we discuss the implications for host-microbial associations and antibacterial therapy.
Under hyperglycaemic conditions non-enzymatic glycation of proteins gives rise to advanced glycation end products (AGEs). The AGEs thus formed generate free radicals, which foster the development of diabetes and its associated complications. Inhibition of glycation is expected to play a role in controlling diabetes. Plant derived antioxidants like cinnamic acid (CA) are known for limiting AGE formation, however, the mechanism involved is poorly understood. Therefore, we aimed to investigate the possible mechanism of inhibition of AGEs formation by CA through various experimental approaches. Glycation of HSA was achieved by incubating the reaction mixture with glucose for 30 days at 37 °C. The protein samples were tested for levels of free lysine & thiol groups, carbonyl content and reactive oxygen species (ROS). Interaction between CA and HSA was also studied through various biophysical techniques. Thermodynamic studies showed a strong exothermic interaction between CA and HSA. The positive value of TΔS° and negative value of ΔH° indicates that the HSA–CA complex is mainly stabilized by a hydrophobic interaction and hydrogen bond. Further, molecular docking reveals that CA binds to HSA subdomain IIA (Sudlow's site I) with a binding energy of −7.0 kcal mol−1, nearly the same as obtained in isothermal titration calorimetry (ITC) and fluorescence spectroscopy. The results of various spectroscopic techniques along with molecular docking and examination of many biomarkers highlights the role of CA in preventing disease progression.
A parallel message-passing implementation of a molecular dynamics (MD) program that is useful for bio(macro)molecules in aqueous environment is described. The software has been developed for a custom-designed 32-processor ring GROMACS (GROningen MAchine for Chemical Simulation) with communication to and from left and right neighbours, but can run on any parallel system onto which a a ring of processors can be mapped and which supports PVM-like block send and receive calls. The GROMACS software consists of a preprocessor, a parallel MD and energy minimization program that can use an arbitrary number of processors (including one), an optional monitor, and several analysis tools. The programs are written in ANSI C and available by ftp (information: gromacs@chem.rug.nl). The functionality is based on the GROMOS (GROningen MOlecular Simulation) package (van Gunsteren and Berendsen, 1987; BIOMOS B.V., Nijenborgh 4, 9747 AG Groningen). Conversion programs between GROMOS and GROMACS formats are included.
Serratia marcescens is an opportunistic turned obligate pathogen frequently associated with urinary tract infections (UTI) and are multidrug resistant at most instances. Quorum sensing (QS) system, a population dependent global regulatory system, controls the pathogenesis machinery of S. marcescens as it does in other pathogens. In the present study, methanol extract of a common herb and spice, Anethum graveolens (AGME) was assessed for its anti-QS potential against the clinical isolate of S. marcescens. AGME notably reduced the biofilm formation and QS dependent virulence factors production in a concentration dependent manner (64-1024 μg mL(-1)). The light and confocal microscopic images clearly evidenced the antibiofilm activity of AGME (256 μg mL(-1)) at its minimal biofilm inhibitory concentration (MBIC). Besides, in support of biochemical assays, the expression analysis of QS regulated genes fimC, bsmA and flhD which are crucial for initial adhesion and motility, confirmed their down regulation upon exposure to AGME. LC-MS analysis of AGME revealed 3-O- methyl ellagic acid (3-O-ME) as one of its active principles having nearly similar antibiofilm activity and a reduced inhibition of prodigiosin (27%) and protease (15%) compared to AGME (prodigiosin (47%) and protease (50%)). UFLC analysis revealed that 0.355 mg g(-1) of 3- O- ME was present in the AGME. AGME and the 3-O-ME significantly interfered the QS system of a QS model strain S. marcescens MG1 and its mutant S. marcescens MG44 which in turn corroborates the anti-QS mechanism of AGME.
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Phenolic phytochemicals include flavonoids, phenolic acids, tannins, lignans, coumarins, quinones, xanthones, cucurmin, and several other plant compounds owing hydroxyl group bonded directly to an aromatic hydrocarbon group. In plants, they play a variety of protective effects against abiotic stresses like UV light or biotic stresses such as predator and pathogen attacks. This role is exploited by humans to treat several ailments including bacterial, fungal, protozoal and viral infections, inflammation, cancer, and diabetes. Numbers of them are known to display direct protection on cells or organs in humans and animals. In contrast, some of others rather have harmful or toxic effects. In this chapter, the synopsis of both protective and harmful effects of phenolics identified in African plants is provided. Emphasis is made on the potential toxic effects of chamuvaritin, gossypol, plumbagin, and scopoletin and the protective roles of catechin, epigallocatechin gallate, genistein, kaempferol, morin, naringenin, quercetin, resveratrol, and rutin.
Molecular Mechanics – Poisson Boltzmann Surface Area (MM-PBSA), a method to estimate interaction free energies, has been increasingly used in the study of bio-molecular interactions. Recently, this method has also been applied as a scoring function in computational drug design. Here a new tool g_mmpbsa, which implements the MM-PBSA approach using subroutines written in-house or sourced from the GROMACS and APBS packages is described. g_mmpbsa was developed as part of the Open Source Drug Discovery (OSDD) consortium. Its aim is to integrate high-throughput MD simulations with binding energy calculations. The tool provides options to select alternative atomic radii and different non-polar solvation models including models based on the solvent accessible surface area (SASA), solvent accessible volume (SAV) and a model which contains both repulsive (SASA-SAV) and attractive components (described using a Weeks-Chandler-Andersen like integral method). We showcase the effectiveness of the tool by comparing the calculated interaction energy of 37 structurally diverse HIV-1 protease inhibitor complexes with their experimental binding free energies. The effect of varying several combinations of input parameters such as atomic radii, dielectric constant, grid resolution, solute-solvent dielectric boundary definition and non-polar models was investigated. g_mmpbsa can also be used to estimate the energy contribution per residue to the binding energy. It has been used to identify those residues in HIV-1 protease that are most critical for binding a range of inhibitors.
AutoDock Vina, a new program for molecular docking and virtual screening, is presented. AutoDock Vina achieves an approximately two orders of magnitude speed-up compared with the molecular docking software previously developed in our lab (AutoDock 4), while also significantly improving the accuracy of the binding mode predictions, judging by our tests on the training set used in AutoDock 4 development. Further speed-up is achieved from parallelism, by using multithreading on multicore machines. AutoDock Vina automatically calculates the grid maps and clusters the results in a way transparent to the user.
Quorum sensing inhibitory (QSI) activity of common South Indian spices and vegetables were evaluated using the bacterial model Chromobacterium violaceum. Among the 22 samples tested the QSI compound present in the methanolic extract of Cuminum cyminum at 2 mg/ml inhibited violacein production in C. violaceum. Further, the outcome of the present investigation reveals that C. cyminum extract strongly interferes with acyl homoserine lactone (AHL) regulated physiological functions coupled with biofilm formation such as flagellar motility and exopolysaccharides (EPS) production. It promotes the loosening of biofilm architecture and powerfully inhibits in vitro biofilm formation in Pseudomonas aeruginosa PAO1, Proteus mirabilis and Serratia marcescens at sub-MIC levels. The result of molecular docking analysis attributes the QSI activity exhibited by C. cyminum to methyl eugenol (ME). The ability of ME to interfere with quorum sensing (QS) systems of various Gram-negative bacterial pathogens comprising diverse AHL molecules was also assessed and ME was found to reduce the AHL dependent production of violacein, bioluminescence and biofilm formation.
Purpose:
Damage caused by an organism during infection is attributed to production of virulence factors. Different virulence factors produced by the organism contribute to its pathogenicity, individually. During infectious conditions, role of virulence factors produced by the pathogen is different, depending upon the site of involvement. Pseudomonas aeruginosa is an opportunistic nosocomial pathogen known to cause infections of the respiratory tract, burn wound, urinary tract and eye. Importance of virulence factors produced by P. Aeruginosa during infections such as keratitis, burn wound and respiratory tract is known. The present study was designed to understand the importance of different virulence factors of P. aeruginosa in urinary tract infection in vivo.
Materials and methods:
An ascending urinary tract infection model was established in mice using standard parent strain PAO1 and its isogenic mutant, JP2. Mice were sacrificed at different time intervals and renal tissue homogenates were used for estimation of renal bacterial load and virulence factors.
Results:
Both parent and mutant strains were able to reach the renal tissue. PAO 1 PAO1 was isolated from renal tissue till day 5 post-infection. However, the mutant strain was unable to colonise the renal tissue. Failure of mutant strain to colonise was attributed to its inability to produce protease, elastase and rhamnolipid.
Conclusion:
This study suggests that protease, elastase and rhamnolipid contribute to pathogenesis and survival of P. aeruginosa during urinary tract infection.
Excessive and indiscriminate use of antibiotics to treat bacterial infections has lead to the emergence of multiple drug resistant strains. Most infectious diseases are caused by bacteria which proliferate within quorum sensing (QS) mediated biofilms. Efforts to disrupt biofilms have enabled the identification of bioactive molecules produced by prokaryotes and eukaryotes. These molecules act primarily by quenching the QS system. The phenomenon is also termed as quorum quenching (QQ). In addition, synthetic compounds have also been found to be effective in QQ. This review focuses primarily on natural and synthetic quorum sensing inhibitors (QSIs) with the potential for treating bacterial infections. It has been opined that the most versatile prokaryotes to produce QSI are likely to be those, which are generally regarded as safe. Among the eukaryotes, certain legumes and traditional medicinal plants are likely to act as QSIs. Such findings are likely to lead to efficient treatments with much lower doses of drugs especially antibiotics than required at present.
Pseudomonas aeruginosa (PA) uses several virulence factors to establish chronic respiratory infections in bronchiectasis, chronic obstructive pulmonary disease, and cystic fibrosis (CF) patients. One of its toxins, pyocyanin (PYO), is a redox-active pigment that is required for full virulence in animal models and has been detected in patients' airway secretions. PYO promotes virulence by interfering with several cellular functions in host cells including electron transport, cellular respiration, energy metabolism, gene expression, and innate immune mechanisms. This review summarizes recent advances in PYO biology with special attention to current views on its role in human airway infections and on its interactions with the first line of our airway defense, the respiratory epithelium.
A parallel message-passing implementation of a molecular dynamics (MD) program that is useful for bio(macro)molecules in aqueous environment is described. The software has been developed for a custom-designed 32-processor ring GROMACS (GROningen MAchine for Chemical Simulation) with communication to and from left and right neighbours, but can run on any parallel system onto which a a ring of processors can be mapped and which supports PVM-like block send and receive calls. The GROMACS software consists of a preprocessor, a parallel MD and energy minimization program that can use an arbitrary number of processors (including one), an optional monitor, and several analysis tools. The programs are written in ANSI C and available by ftp (information: [email protected]
/* */). The functionality is based on the GROMOS (GROningen MOlecular Simulation) package (van Gunsteren and Berendsen, 1987; BIOMOS B.V., Nijenborgh 4, 9747 AG Groningen). Conversion programs between GROMOS and GROMACS formats are included. The MD program can handle rectangular periodic boundary conditions with temperature and pressure scaling. The interactions that can be handled without modification are variable non-bonded pair interactions with Coulomb and Lennard-Jones or Buckingham potentials, using a twin-range cut-off based on charge groups, and fixed bonded interactions of either harmonic or constraint type for bonds and bond angles and either periodic or cosine power series interactions for dihedral angles. Special forces can be added to groups of particles (for non-equilibrium dynamics or for position restraining) or between particles (for distance restraints). The parallelism is based on particle decomposition. Interprocessor communication is largely limited to position and force distribution over the ring once per time step.
Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogen responsible for a wide range of infections in immunocompromized individuals and is a leading cause of mortality in cystic fibrosis patients. A number of secreted virulence factors, including various proteolytic enzymes, contribute to the establishment and maintenance of Pseudomonas infection. One such is LasA, an M23 metallopeptidase related to autolytic glycylglycine endopeptidases such as Staphylococcus aureus lysostaphin and LytM, and to DD-endopeptidases involved in entry of bacteriophage to host bacteria. LasA is implicated in a range of processes related to Pseudomonas virulence, including stimulating ectodomain shedding of the cell surface heparan sulphate proteoglycan syndecan-1 and elastin degradation in connective tissue. Here we present crystal structures of active LasA as a complex with tartrate and in the uncomplexed form. While the overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Using these structures as a starting point, we propose a model for substrate binding by LasA that explains its activity against a wider range of substrates than those used by related lytic enzymes, and offer a catalytic mechanism for M23 metallopeptidases consistent with available structural and mutagenesis data. Our results highlight how LasA is a structurally distinct member of this endopeptidase family, consistent with its activity against a wider range of substrates and with its multiple roles in Pseudomonas virulence.
Plumbagin, a naphtoquinone present in the roots of Plumbago zeylanica, has been reported to have many beneficial effects such as antibacterial, antifungal, anticancer, antimutagenic and antioxidant effects, but this compound has also been reported to have many side effects. Given the wide use of P. zeylanica in traditional medicine and the various potential therapeutic uses of plumbagin, the present study was carried out to further elucidate the potential genotoxicity and antigenotoxicity of plumbagin in mouse lymphoma L5178Y cells, using the comet assay. Without affecting the cell viability, plumbagin itself was found to induce significant DNA damage at concentrations as low as 0.25 ng/ml. When the cells were exposed to non-DNA damaging concentrations of plumbagin, together with NQNO (known to interact with DNA in many different ways) or catechol (known to induce oxidative DNA damage), plumbagin was found to significantly reduce the catechol-induced DNA damage, but to be without protective effect against the NQNO-induced damage. The fact that non-DNA damaging concentrations of plumbagin diminished the DNA damage induced by catechol, provides further support for the idea that plumbagin may act as an antioxidative agent at low concentrations.
This paper reports the antimutagenic activity of plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) against certain known chemical mutagens in a standard mutagenicity test system of Ames using S. typhimurium strains. Plumbagin by itself did not show any mutagenic effect, whereas it reduced significantly the mutagenic effect of 4-nitrophenylene diammine, phenyl hydrazine and sodium azide in test strains of S. typhimurium, suggesting that plumbagin possessed antimutagenic activity.
Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.
A combination of the siderophores produced by Pseudomonas aeruginosa, pyochelin and pyoverdin, dramatically stimulates the growth of this bacterium in medium containing human transferrin. The amount of growth stimulation observed when each siderophore was added alone was only slightly less than the amount observed with the combination. Siderophore-defective mutants of strain PAO1 were isolated to test the effects of siderophore production on growth in transferrin and human serum. The pyoverdin-proficient (Pvd+), pyochelin-deficient (Pch-) strain (IA5) grows just as well as the parent (PAO1), which produces both siderophores. On the other hand, the Pvd- Pch+ strain (211-5) has severely retarded growth, similar to that demonstrated by a mutant lacking production of both siderophores (IA1), but has an accelerated log phase compared with strain IA1 at the later stages of the growth curve. However, the Pvd- Pch+ strain (211-5) had no observable advantage over the Pvd- Pch- strain, IA1, during incubation in human serum. The inability of P. aeruginosa strains to produce pyochelin in glucose-minimal medium may explain the poor growth of 211-5 in this medium and in human serum. The 211-5 strain grows much better than the IA1 strain in the medium that allows pyochelin synthesis, but it still does not grow as well as the Pvd+ Pch- strain (IA5). Therefore, pyoverdin appears to be the most important siderophore for growth in human serum.
Pseudomonas aeruginosa elastase is a zinc metalloendopeptidase, probably responsible for the tissue destruction observed during infections with this organism. The elastase of a virulent Pseudomonas aeruginosa strain (Habs serotype 1) was isolated and found to have a molecular weight of 35,000; it readily degraded elastin and cartilage proteoglycans. A series of amino acid and peptide derivatives containing the metal-chelating moieties hydroxamate, phosphoryl, or thiol were synthesized and tested as potential inhibitors of the enzyme. Inhibition constants (K(i)s) for the compounds were determined with the chromophoric substrate furylacryloyl-glycyl-l-leucyl-l-alanine. The hydroxamic acid derivatives of benzyloxycarbonyl-glycine, benzyloxycarbonyl-l-leucine and benzyloxycarbonyl-l-phenylalanine had inhibition constants in the range of 11 to 28 muM. The 2-mercaptoacetyl derivatives of l-leucyl-d-phenylalanine and l-leucyl-l-phenylalanine had K(i) values of 34 and 1.5 muM, respectively, demonstrating the stereospecificity of the inhibition. The most potent inhibitors tested were 2- mercaptoacetyl-l-phenylalanyl-l-leucine and phosphoryl-l-leucyl-l-phenylala-nine (K(i) = 0.2 muM). Similar compounds lacking the metal-chelating moiety were about 3 orders of magnitude poorer inhibitors. When the inhibition of the enzyme activity towards azocasein, elastin, or cartilage was examined, inhibitor concentrations approximately 50-fold higher than the respective K(i)s were required to obtain 60 to 90% inhibition. Virtually complete inhibition was achieved with these substrates at inhibitor concentrations 500-fold higher than the respective K(i)s (0.1 to 14 mM). Although, 2-mercaptoacetyl-l-phenylalanyl-l-leucine and phosphoryl-l-leucyl-l-phenylalanine exhibited the same affinity to the enzyme, the latter was inferior in inhibiting cartilage proteoglycan degradation. 2-Mercaptoacetyl-l-phenylalanyl-l-leucine represents a class of potent elastase inhibitors that might prove useful in the management of P. aeruginosa infections.