Biofilm inhibition/eradication: exploring strategies and confronting challenges in combatting biofilm.

Biofilms are complex communities of microorganisms enclosed in a self-produced extracellular matrix, posing a significant threat to different sectors, including healthcare and industry. This review provides an overview of the challenges faced due to biofilm formation and different novel strategies that can combat biofilm formation. Bacteria inside the biofilm exhibit increased resistance against different antimicrobial agents, including conventional antibiotics, which can lead to severe problems in livestock and animals, including humans. In addition, biofilm formation also imposes heavy economic pressure on industries. Hence it becomes necessary to explore newer alternatives to eradicate biofilms effectively without applying selection pressure on the bacteria. Excessive usage of antibiotics may also lead to an increase in the number of resistant strains as bacteria employ an advanced antimicrobial resistance mechanism. This review provides insight into multifaceted technologies like quorum sensing inhibition, enzymes, antimicrobial peptides, bacteriophage, phytocompounds, and nanotechnology to neutralize biofilms without developing antimicrobial resistance (AMR). Furthermore, it will pave the way for developing newer therapeutic agents to deal with biofilms more efficiently.

[Research progress of traditional Chinese medicines as quorum sensing inhibitors in collaboration with antibiotics to inhibit drug-resistant bacteria].

Quorum sensing system regulates the expression of genes related to bacterial growth, metabolism and other behaviors by sensing bacterial density, and controls the unified action of the entire bacterial population. This mechanism can ensure the normal secretion of bacterial metabolites and the stability of the biofilm microenvironment, providing protection for the formation of biofilms and the normal growth and reproduction of bacteria. Traditional Chinese medicine, capable of quorum sensing inhibition, can inhibit the formation of bacterial biofilms, reduce bacterial resistance, and enhance the anti-infection ability of antibiotics when combined with antibiotics. In recent years, the combination of traditional Chinese and Western medicine in the treatment of drug-resistant bacterial infections has become a research hotspot. Starting with the associations between quorum sensing, biofilm and drug-resistant bacteria, this paper reviews the relevant studies about the combined application of traditional Chinese medicines as quorum sensing inhibitors with antibiotics in the treatment of drug-resistant bacteria. This review is expected to provide ideas for the development of new clinical treatment methods and novel anti-infection drugs.

Cue-driven microbial cooperation and communication: evolving quorum sensing with honest signaling.

BACKGROUND: Quorum sensing (QS) is the ability of microorganisms to assess local clonal density by measuring the extracellular concentration of signal molecules that they produce and excrete. QS is also the only known way of bacterial communication that supports the coordination of within-clone cooperative actions requiring a certain threshold density of cooperating cells. Cooperation aided by QS communication is sensitive to cheating in two different ways: laggards may benefit from not investing in cooperation but enjoying the benefit provided by their cooperating neighbors, whereas Liars explicitly promise cooperation but fail to do so, thereby convincing potential cooperating neighbors to help them, for almost free. Given this double vulnerability to cheats, it is not trivial why QS-supported cooperation is so widespread among prokaryotes. RESULTS: We investigated the evolutionary dynamics of QS in populations of cooperators for whom the QS signal is an inevitable side effect of producing the public good itself (cue-based QS). Using spatially explicit agent-based lattice simulations of QS-aided threshold cooperation (whereby cooperation is effective only above a critical cumulative level of contributions) and three different (analytical and numerical) approximations of the lattice model, we explored the dynamics of QS-aided threshold cooperation under a feasible range of parameter values. We demonstrate three major advantages of cue-driven cooperation. First, laggards cannot wipe out cooperation under a wide range of reasonable environmental conditions, in spite of an unconstrained possibility to mutate to cheating; in fact, cooperators may even exclude laggards at high cooperation thresholds. Second, lying almost never pays off, if the signal is an inevitable byproduct (i.e., the cue) of cooperation; even very cheap fake signals are selected against. And thirdly, QS is most useful if local cooperator densities are the least predictable, i.e., if their lattice-wise mean is close to the cooperation threshold with a substantial variance. CONCLUSIONS: Comparing the results of the four different modeling approaches indicates that cue-driven threshold cooperation may be a viable evolutionary strategy for microbes that cannot keep track of past behavior of their potential cooperating partners, in spatially viscous and in well-mixed environments alike. Our model can be seen as a version of the famous greenbeard effect, where greenbeards coexist with defectors in a evolutionarily stable polymorphism. Such polymorphism is maintained by the condition-dependent trade-offs of signal production which are characteristic of cue-based QS.

Design, Synthesis, and Biological Evaluation of 2-Phenoxyalkylhydrazide Benzoxazole Derivatives as Quorum Sensing Inhibitors with Strong Antibiofilm Effect.

With the increasing problem of bacterial resistance to traditional antibiotics, there is an urgent need for new antibacterial agents with novel mechanisms to treat infections caused by drug-resistant bacteria. In this paper, we designed and synthesized 2-phenoxyalkylhydrazide benzoxazole derivatives and evaluated their quorum sensing inhibition activity. Among them, 26c at a concentration of 102.4 µg/mL not only inhibited the production of pyocyanin and rhamnolipid by 45.6% and 38.3%, respectively, but also suppressed 76.6% of biofilm production at 32 µg/mL. In addition, 26c did not affect bacterial growth, but in a mouse model infected with P. aeruginosa PAO1, it could help ciprofloxacin effectively eliminate the living bacteria. In the targeting experiment, 26c could inhibit the fluorescence intensity of PAO1-lasB-gfp and PAO1-pqsA-gfp in a concentration-dependent manner, indicating that the compound acts on the quorum sensing system. Overall, 26c is worthy of further investigation as a quorum sensing inhibitor with strong antibiofilm effect.

Effects of elevated levels of intracellular nitric oxide on Pseudomonas aeruginosa biofilm in chronic skin wound and slow-killing infection models

Nitric oxide (NO), produced through the denitrification pathway, regulates biofilm dynamics through the quorum sensing system in Pseudomonas aeruginosa. NO stimulates P. aeruginosa biofilm dispersal by enhancing phosphodiesterase activity to decrease cyclic di-GMP levels. In a chronic skin wound model containing a mature biofilm, the gene expression of nirS, encoding nitrite reductase to produce NO, was low, leading to reduced intracellular NO levels. Although low-dose NO induces biofilm dispersion, it is unknown whether it influences the formation of P. aeruginosa biofilms in chronic skin wounds. In this study, a P. aeruginosa PAO1 strain with overexpressed nirS was established to investigate NO effects on P. aeruginosa biofilm formation in an ex vivo chronic skin wound model and unravel the underlying molecular mechanisms. Elevated intracellular NO levels altered the biofilm structure in the wound model by inhibiting the expression of quorum sensing–related genes, which was different from an in vitro model. In Caenorhabditis elegans as a slow-killing infection model, elevated intracellular NO levels increased worms’ lifespan by 18%. Worms that fed on the nirS-overexpressed PAO1 strain for 4 h had complete tissue, whereas worms that fed on empty plasmid–containing PAO1 had biofilms on their body, causing severe damage to the head and tail. Thus, elevated intracellular NO levels can inhibit P. aeruginosa biofilm growth in chronic skin wounds and reduce pathogenicity to the host. Targeting NO is a potential approach to control biofilm growth in chronic skin wounds wherein P. aeruginosa biofilms are a persistent problem. (AU)

A novel photoelectrochemical self-screening aptamer biosensor based on CAU-17-derived Bi2WO6/Bi2S3 for rapid detection of quorum sensing signal molecules.

Quorum sensing signal molecule is an important biomarker released by some microorganisms, which can regulate the adhesion and aggregation of marine microorganisms on the surface of engineering facilities. Thus, it is significant to exploit a convenient method that can effectively monitor the formation and development of marine biofouling. In this work, an advanced photoelectrochemical (PEC) aptamer biosensing platform was established and firstly applied for the rapid and ultrasensitive determination of N-(3-Oxodecanoyl)-l-homoserine lactone (3-O-C10-HL) released from marine fouling microorganism Ponticoccus sp. PD-2. The visible-light-driven Bi2WO6/Bi2S3 heterojunction derived from metal-organic frameworks (MOFs) CAU-17 and self-screened aptamer were employed as the photoactive materials and bioidentification elements, respectively. Appropriate amount of MoS2 quantum dots (QDs) conjugated with single-stranded DNA were introduced by hybridization to enhance the photocurrent response of the PEC biosensor. The self-screening aptamer can specifically recognize 3-O-C10-HL, accompanied by increasing the steric hindrance and forcing MoS2 QDs to leave the electrode surface, resulting in an obvious reduction of photocurrent and achieving a dual-inhibition signal amplification effect. Under the optimized conditions, the photocurrent response of PEC aptasensor was linear with 3-O-C10-HL concentration from 1 nM to 10 µM, and the detection limit was as low as 0.26 nM. The detection strategy also showed a high reproducibility, superior specificity and good stability. This work not only provides a simple, rapid and ultrasensitive PEC aptamer biosensing strategy for monitoring quorum sensing signal molecules in marine biofouling, but also broadens the application of MOFs-based heterojunctions in PEC sensors.

Thymoquinone is a natural antibiofilm and pathogenicity attenuating agent in Pseudomonas aeruginosa.

Pseudomonas aeruginosa belongs to the critical pathogens that represent a global public health problem due to their high rate of resistance as listed by WHO. P. aeruginosa can result in many nosocomial infections especially in individuals with compromised immune systems. Attenuating virulence factors by interference with quorum sensing (QS) systems is a promising approach to treat P. aeruginosa-resistant infections. Thymoquinone is a natural compound isolated from Nigella sativa (black seed) essential oil. In this study, the minimum inhibitory concentration of thymoquinone was detected followed by investigating the antibiofilm and antivirulence activities of the subinhibitory concentration of thymoquinone against P. aeruginosa PAO1. The effect of thymoquinone on the expression of QS genes was assessed by quantitative real-time PCR, and the protective effect of thymoquinone against the pathogenesis of PAO1 in mice was detected by the mouse survival test. Thymoquinone significantly inhibited biofilm, pyocyanin, protease activity, and swarming motility. At the molecular level, thymoquinone markedly downregulated QS genes lasI, lasR, rhlI, and rhlR. Moreover, thymoquinone could protect mice from the pathologic effects of P. aeruginosa increasing mouse survival from 20% to 100%. In conclusion, thymoquinone is a promising natural agent that can be used as an adjunct therapeutic agent with antibiotics to attenuate the pathogenicity of P. aeruginosa.

Study on the inhibition activity and mechanism of Tanreqing against Klebsiella pneumoniae biofilm formation in vitro and in vivo.

Objective: To evaluate the antibacterial effect of Tanreqing (TRQ) against K. pneumoniae and its inhibition activity on bacterial biofilm formation in vitro and in vivo, and to explore the mechanism of the inhibitory effects of TRQ on K. pneumoniae biofilm formation. Methods: An in vitro biofilm model of K. pneumoniae was established, and the impact of TRQ on biofilm formation was evaluated using crystal violet staining and scanning electron microscopy (SEM). Furthermore, the clearance effect of TRQ against K. pneumoniae in the biofilm was assessed using the viable plate counting method; q-RT PCR was used to evaluate the inhibitory effect of different concentrations of TRQ on the expression of biofilm-related genes in Klebsiella pneumoniae; The activity of quorum sensing signal molecule AI-2 was detected by Vibrio harveyi bioluminescence assay; Meanwhile, a guinea pig lung infection model of Klebsiella pneumoniae was constructed, and after treated with drugs, pathological analysis of lung tissue and determination of bacterial load in lung tissue were performed. The treatment groups included TRQ group, imipenem(IPM) group, TRQ+IPM group, and sterile saline group as the control. Results: The formation of K. pneumoniae biofilm was significantly inhibited by TRQ in vitro experiments. Furthermore, when combined with IPM, the clearance of K. pneumoniae in the biofilm was notably increased compared to the TRQ group and IPM group alone. q-RT PCR analysis revealed that TRQ down-regulated the expression of genes related to biofilm formation in K. pneumoniae, specifically luxS, wbbm, wzm, and lsrK, and also inhibited the activity of AI-2 molecules in the bacterium. In vivo experiments demonstrated that TRQ effectively treated guinea pig lung infections, resulting in reduced lung inflammation. Additionally, when combined with IPM, there was a significant reduction in the bacterial load in lung tissue. Conclusion: TRQ as a potential therapeutic agent plays a great role in the treatment of K. pneumoniae infections, particularly in combination with conventional antibiotics. And TRQ can enhanced the clearance effect on the bacterium by inhibiting the K. pneumoniae biofilm formation, which provided experimental evidence in support of clinical treatment of TRQ against K. pneumoniae infections.

Glycol Chitosan-Poly(lactic acid) Conjugate Nanoparticles Encapsulating Ciprofloxacin: A Mucoadhesive, Antiquorum-Sensing, and Biofilm-Disrupting Treatment Modality for Bacterial Keratitis.

Bacterial keratitis (BK) causes visual morbidity/blindness if not treated effectively. Here, ciprofloxacin (CIP)-loaded nanoparticles (NPs) using glycol chitosan (GC) and poly(lactic acid) (PLA) conjugate at three different ratios (CIP@GC(PLA) NPs (1:1,5,15)) were fabricated. CIP@GC(PLA) NPs (1:1) were more effective than other tested ratios, indicating the importance of optimal hydrophobic/hydrophilic balance for corneal penetration and preventing bacterial invasion. The CIP@GC(PLA) (NPs) (1:1) realized the highest association with human corneal epithelial cells, which were nonirritant to the hen's egg-chorioallantoic membrane test (HET-CAM test) and demonstrated significant antibacterial response in the in vitro minimum inhibitory, bactericidal, live-dead cells, zone of inhibition, and biofilm inhibition assays against the keratitis-inducing pathogen Pseudomonas aeruginosa. The antiquorum sensing activity of GC has been explored for the first time. The NPs disrupted the bacterial quorum sensing by inhibiting the production of virulence factors, including acyl homoserine lactones, pyocyanin, and motility, and caused significant downregulation of quorum sensing associated genes. In the in vivo studies, CIP@GC(PLA) NPs (1:1) displayed ocular retention in vivo (∼6 h) and decreased the opacity and the bacterial load effectively. Overall, the CIP@GC(PLA) NP (1:1) is a biofilm-disrupting antiquorum sensing treatment regimen with clinical translation potential in BK.

Research Progress on the Combination of Quorum-Sensing Inhibitors and Antibiotics against Bacterial Resistance.

Bacterial virulence factors and biofilm development can be controlled by the quorum-sensing (QS) system, which is also intimately linked to antibiotic resistance in bacteria. In previous studies, many researchers found that quorum-sensing inhibitors (QSIs) can affect the development of bacterial biofilms and prevent the synthesis of many virulence factors. However, QSIs alone have a limited ability to suppress bacteria. Fortunately, when QSIs are combined with antibiotics, they have a better therapeutic effect, and it has even been demonstrated that the two together have a synergistic antibacterial effect, which not only ensures bactericidal efficiency but also avoids the resistance caused by excessive use of antibiotics. In addition, some progress has been made through in vivo studies on the combination of QSIs and antibiotics. This article mainly expounds on the specific effect of QSIs combined with antibiotics on bacteria and the combined antibacterial mechanism of some QSIs and antibiotics. These studies will provide new strategies and means for the clinical treatment of bacterial infections in the future.

Quenching of quorum sensing in multi-drug resistant Pseudomonas aeruginosa: insights on halo-bacterial metabolites and gamma irradiation as channels inhibitors.

BACKGROUND: Anti-virulence therapy is a promising strategy to treat multi-drug resistant (MDR) pathogens. Pseudomonas aeruginosa is a potent opportunistic pathogen because of an array of virulence factors that are regulated by quorum sensing systems. METHODS: The virulence features of four multi-drug resistant P. aeruginosa strains were investigated upon exposure to the sub-lethal dose of gamma rays (1 kGy), and sub-inhibitory concentrations of bioactive metabolites recovered from local halophilic strains in comparison to control. Then, the gene expression of AHL-mediated quorum sensing systems (las/rhl) was quantitatively determined in treated and untreated groups by real-time PCR. RESULTS: The bioactive metabolites recovered from halophilic strains previously isolated from saline ecosystems were identified as Halomonas cupida (Halo-Rt1), H. elongate (Halo-Rt2), Vigibacillus natechei (Halo-Rt3), Sediminibacillus terrae (Halo-Rt4) and H. almeriensis (Halo-Rt5). Results revealed that both gamma irradiation and bioactive metabolites significantly reduced the virulence factors of the tested MDR strains. The bioactive metabolites showed a maximum efficiency for inhibiting biofilm formation and rhamnolipids production whereas the gamma irradiation succeeded in decreasing other virulence factors to lower levels in comparison to control. Quantitative-PCR results showed that AHL-mediated quorum sensing systems (las/rhl) in P. aeruginosa strains were downregulated either by halo-bacterial metabolites or gamma irradiation in all treatments except the upregulation of both lasI internal gene and rhlR intact gene in P. aeruginosa NCR-RT3 and both rhlI internal gene and rhlR intact gene in P. aeruginosa U3 by nearly two folds or more upon exposure to gamma irradiation. The most potent result was observed in the expression of lasI internal gene that was downregulated by more than ninety folds in P. aeruginosa NCR-RT2 after treatment with metabolites of S. terrae (Halo-Rt4). Analyzing metabolites recovered from H. cupida (Halo-Rt1) and H. elongate (Halo-Rt2) using LC-ESI-MS/MS revealed many chemical compounds that have quorum quenching properties including glabrol, 5,8-dimethoxyquinoline-2-carbaldehyde, linoleoyl ethanolamide, agelasine, penigequinolones derivatives, berberine, tetracosanoic acid, and liquidambaric lactone in the former halophile and phloretin, lycoctonine, fucoxanthin, and crassicauline A in the latter one. CONCLUSION: QS inhibitors can significantly reduce the pathogenicity of MDR P. aeruginosa strains; and thus can be an effective and successful strategy for treating antibiotic resistant traits.

Decoding Microcystis aeruginosa quorum sensing through AHL-mediated transcriptomic molecular regulation mechanisms.

Acyl-homoserine lactone (AHL) serves as a key signaling molecule for quorum sensing (QS) in bacteria. QS-related genes and physiological processes in Microcystis aeruginosa remain elusive. In this study, we elucidated the regulatory role of AHL-mediated QS in M. aeruginosa. Using AHL activity extract and transcriptomic analysis, we revealed significant effects of the AHL on growth and photosynthesis. AHL significantly increased chlorophyll a (Chl-a) content and accelerated photosynthetic rate thereby promoting growth. Transcriptome analysis revealed that AHL stimulated the up-regulation of photosynthesis-related genes (apcABF, petE, psaBFK, psbUV, etc.) as well as nitrogen metabolism and ribosomal metabolism. In addition, AHL-regulated pathways are associated with lipopolysaccharide and phenazine synthesis. Our findings deepen the understanding of the QS system in M. aeruginosa and are important for gaining insights into the role of QS in Microcystis bloom formation. It also provides new insights into the prevalence of M. aeruginosa in water blooms.

Laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS) demonstrates a direct impact of hypochlorous acid stress on PQS-mediated quorum sensing in Pseudomonas aeruginosa.

To establish infections in human hosts, Pseudomonas aeruginosa must overcome innate immune-generated oxidative stress, such as the hypochlorous acid (HOCl) produced by neutrophils. We set out to find specific biomarkers of oxidative stress through the development of a protocol for the metabolic profiling of P. aeruginosa cultures grown in the presence of different oxidants using a novel ionization technique for mass spectrometry, laser desorption rapid evaporative ionization mass spectrometry (LD-REIMS). We demonstrated the ability of LD-REIMS to classify samples as untreated or treated with a specific oxidant with 100% accuracy and identified a panel of 54 metabolites with significantly altered concentrations after exposure to one or more of the oxidants. Key metabolic changes were conserved in P. aeruginosa clinical strains isolated from patients with cystic fibrosis lung infections. These data demonstrated that HOCl stress impacted the Pseudomonas quinolone signal (PQS) quorum sensing system. Ten 2-alkyl-4-quinolones (AHQs) associated with the PQS system were significantly lower in concentration in HOCl-stressed P. aeruginosa cultures, including 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), the most active signal molecule of the PQS system. The PQS system regulates the production of virulence factors, including pyocyanin and elastase, and their levels were markedly affected by HOCl stress. No pyocyanin was detectable and elastase concentrations were reduced by more than 75% in cultures grown with sub-lethal concentrations of HOCl, suggesting that this neutrophil-derived oxidant may disrupt the ability of P. aeruginosa to establish infections through interference with production of PQS-associated virulence factors. IMPORTANCE: This work demonstrates that a high-throughput ambient ionization mass spectrometry method can be used successfully to study a bacterial stress response. Its application to the opportunistic pathogen Pseudomonas aeruginosa led to the identification of specific oxidative stress biomarkers, and demonstrated that hypochlorous acid, an oxidant specifically produced by human neutrophils during infection, affects quorum sensing and reduces production of the virulence factors pyocyanin and elastase. No pyocyanin was detectable and elastase levels were reduced by more than 75% in bacteria grown in the presence of hypochlorous acid. This approach has the potential to be widely applicable to the characterization of the stress responses of bacteria.

Understanding the biofilm development of Acinetobacter baumannii and novel strategies to combat infection.

Acinetobacter baumannii (A. baumannii) is a Gram-negative, nonmotile, and aerobic bacillus emerged as a superbug, due to increasing the possibility of infection and accelerating rates of antimicrobial agents. It is recognized as a nosocomial pathogen due to its ability to form biofilms. These biofilms serve as a defensive barrier, increase antibiotic resistance, and make treatment more difficult. As a result, the current situation necessitates the rapid emergence of novel therapeutic approaches to ensure successful treatment outcomes. This review explores the intricate relationship between biofilm formation and antibiotic resistance in A. baumannii, emphasizing the role of key virulence factors and quorum sensing (QS) mechanisms that will lead to infections and facilitate insight into developing innovative method to control A. baumannii infections. Furthermore, the review article looks into promising approaches for preventing biofilm formation on medically important surfaces and potential therapeutic methods for eliminating preformed biofilms, which can address biofilm-associated A. baumannii infections. Modern advances in emerging therapeutic options such as antimicrobial peptide (AMPs), nanoparticles (NPs), bacteriophage therapy, photodynamic therapy (PDT), and other biofilm inhibitors can assist readers understand the current landscape and future prospects for effectively treating A. baumannii biofilm infections.

D-histidine combated biofilm formation and enhanced the effect of amikacin against Pseudomonas aeruginosa in vitro.

Pseudomonas aeruginosa is an opportunistic gram-negative pathogenic microorganism that poses a significant challenge in clinical treatment. Antibiotics exhibit limited efficacy against mature biofilm, culminating in an increase in the number of antibiotic-resistant strains. Therefore, novel strategies are essential to enhance the effectiveness of antibiotics against Pseudomonas aeruginosa biofilms. D-histidine has been previously identified as a prospective anti-biofilm agent. However, limited attention has been directed towards its impact on Pseudomonas aeruginosa. Therefore, this study was undertaken to explore the effect of D-histidine on Pseudomonas aeruginosa in vitro. Our results demonstrated that D-histidine downregulated the mRNA expression of virulence and quorum sensing (QS)-associated genes in Pseudomonas aeruginosa PAO1 without affecting bacterial growth. Swarming and swimming motility tests revealed that D-histidine significantly reduced the motility and pathogenicity of PAO1. Moreover, crystal violet staining and confocal laser scanning microscopy demonstrated that D-histidine inhibited biofilm formation and triggered the disassembly of mature biofilms. Notably, D-histidine increased the susceptibility of PAO1 to amikacin compared to that in the amikacin-alone group. These findings underscore the efficacy of D-histidine in combating Pseudomonas aeruginosa by reducing biofilm formation and increasing biofilm disassembly. Moreover, the combination of amikacin and D-histidine induced a synergistic effect against Pseudomonas aeruginosa biofilms, suggesting the potential utility of D-histidine as a preventive strategy against biofilm-associated infections caused by Pseudomonas aeruginosa.

Engineering quorum quenching acylases with improved kinetic and biochemical properties.

Many Gram-negative bacteria use N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm-related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to generate these improved properties using the Protein One-Stop Shop Server. The engineering of acylases is complicated by low-throughput enzymatic assays. Alleviating this challenge, we report a time-course kinetic assay for AHL acylases that monitors the real-time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases.

Enzymes with Lactonase Activity against Fungal Quorum Molecules as Effective Antifungals.

Since the growing number of fungi resistant to the fungicides used is becoming a serious threat to human health, animals, and crops, there is a need to find other effective approaches in the eco-friendly suppression of fungal growth. One of the main mechanisms of the development of resistance in fungi, as well as in bacteria, to antimicrobial agents is quorum sensing (QS), in which various lactone-containing compounds participate as signaling molecules. This work aimed to study the effectiveness of action of enzymes exhibiting lactonase activity against fungal signaling molecules. For this, the molecular docking method was used to estimate the interactions between these enzymes and different lactone-containing QS molecules of fungi. The catalytic characteristics of enzymes such as lactonase AiiA, metallo-ß-lactamase NDM-1, and organophosphate hydrolase His6-OPH, selected for wet experiments based on the results of computational modeling, were investigated. QS lactone-containing molecules (butyrolactone I and γ-heptalactone) were involved in the experiments as substrates. Further, the antifungal activity of the enzymes was evaluated against various fungal and yeast cells using bioluminescent ATP-metry. The efficient hydrolysis of γ-heptalactone by all three enzymes and butyrolactone I by His6-OPH was demonstrated for the first time. The high antifungal efficacy of action of AiiA and NDM-1 against most of the tested fungal cells was revealed.

Implementation of a Quorum-Sensing System for Highly Efficient Biosynthesis of Lacto-N-neotetraose in Engineered Escherichia coli MG1655.

Lacto-N-neotetraose (LNnT), a prominent neutral human milk oligosaccharide (HMO), serves as a pivotal structural element in complex HMO biosynthesis. Given its promising health effects for infants, the biosynthesis of LNnT is garnering greater interest. Using a previously engineered strain as a chassis, a highly effective LNnT producer was constructed. First, LNnT synthesis in Escherichia coli MG1655 was achieved by introducing ß1,3-N-acetylglucosaminyltransferase LgtA and ß1,4-galactosyltransferase CpsIaJ, coupled with the optimization of enzyme expression levels using various promoters. Subsequently, ugd underwent disruption, and the galE gene was enhanced by replacing its promoter with PJ23119 or Ptac. Then, a lux-type quorum sensing (QS) system was applied to achieve varied metabolic regulation. Additionally, systematic optimization of the QS promoters was conducted to further improve the LNnT titer in the shake flask. Finally, the extracellular titer of LNnT was 20.33 g/L, accompanied by a productivity of 0.41 g/L/h.

Abiotic Small Molecule Inhibitors and Activators of the LasR Quorum Sensing Receptor in Pseudomonas aeruginosa with Potencies Comparable or Surpassing N-Acyl Homoserine Lactones.

The opportunistic pathogen Pseudomonas aeruginosa controls almost 10% of its genome, including myriad virulence genes, via a cell-to-cell chemical communication system called quorum sensing (QS). Small molecules that either inhibit or activate QS in P. aeruginosa represent useful research tools to study the role of this signaling pathway in infection and interrogate its viability as an antivirulence target. However, despite active research in this area over the past 20+ years, there are relatively few synthetic compounds known to strongly inhibit or activate QS in P. aeruginosa. Most reported QS modulators in this pathogen are of low potency or have structural liabilities that limit their application in biologically relevant environments such as mimics of the native N-acyl l-homoserine lactone (AHL) signals. Here, we report the results of a high-throughput screen for abiotic small molecules that target LasR, a key QS regulator in P. aeruginosa. We screened a 25,000-compound library and discovered four new structural classes of abiotic LasR modulators. These compounds include antagonists that surpass the potency of all known AHL-type compounds and mimetics thereof, along with an agonist with potency approaching that of LasR's native ligand. The novel structures of this compound set, along with their anticipated robust physicochemical profiles, underscore their potential value as probe molecules to interrogate the roles of QS in this formidable pathogen.

Unveiling the trifecta of cyanobacterial quorum sensing: LuxI, LuxR and LuxS as the intricate machinery for harmful algal bloom formation in freshwater ecosystems.

Harmful algal blooms (HABs) are causing significant disruptions in freshwater ecosystems, primarily due to the proliferation of cyanobacteria. These blooms have a widespread impact on various lakes globally, leading to profound environmental and health consequences. Cyanobacteria, with their ability to produce diverse toxins, pose a particular concern as they negatively affect the well-being of humans and animals, exacerbating the situation. Notably, cyanobacteria utilize quorum sensing (QS) as a complex communication mechanism that facilitates coordinated growth and toxin production. QS plays a critical role in regulating the dynamics of HABs. However, recent advances in control and mitigation strategies have shown promising results in effectively managing and reducing the occurrence of HABs. This comprehensive review explores the intricate aspects of cyanobacteria development in freshwater ecosystems, explicitly focusing on deciphering the signaling molecules associated with QS and their corresponding genes. Furthermore, a concise overview of diverse measures implemented to efficiently control and mitigate the spread of these bacteria will be provided, shedding light on the ongoing global efforts to address this urgent environmental issue. By deepening our understanding of the mechanisms driving cyanobacteria growth and developing targeted control strategies, we hope to safeguard freshwater ecosystems and protect the health of humans and animals from the detrimental impacts of HABs.