Design, Synthesis, and Evaluation of New 1H-Benzo[d]imidazole Based PqsR Inhibitors as Adjuvant Therapy for Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor 1 (IC50 3.2 µM in P. aeruginosa PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled PpqsA-lux transcriptional reporter fusion in P. aeruginosa at low submicromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)-quinolones production.

Convergence of resistance and evolutionary responses in Escherichia coli and Salmonella enterica co-inhabiting chicken farms in China.

Sharing of genetic elements among different pathogens and commensals inhabiting same hosts and environments has significant implications for antimicrobial resistance (AMR), especially in settings with high antimicrobial exposure. We analysed 661 Escherichia coli and Salmonella enterica isolates collected within and across hosts and environments, in 10 Chinese chicken farms over 2.5 years using data-mining methods. Most isolates within same hosts possessed the same clinically relevant AMR-carrying mobile genetic elements (plasmids: 70.6%, transposons: 78%), which also showed recent common evolution. Supervised machine learning classifiers revealed known and novel AMR-associated mutations and genes underlying resistance to 28 antimicrobials, primarily associated with resistance in E. coli and susceptibility in S. enterica. Many were essential and affected same metabolic processes in both species, albeit with varying degrees of phylogenetic penetration. Multi-modal strategies are crucial to investigate the interplay of mobilome, resistance and metabolism in cohabiting bacteria, especially in ecological settings where community-driven resistance selection occurs.

RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing.

In its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that considerable cell-to-cell variation in the QS activation state occurs during growth, often resulting in coexisting subpopulations of cells in which QS is active (quorate cells) or inactive (non-quorate cells). Heterogeneity has been observed in the las QS system of the opportunistic pathogen Pseudomonas aeruginosa. However, the molecular mechanisms underlying this phenomenon have not yet been defined. The las QS system consists of an incoherent feedforward loop in which the LasR transcriptional regulator activates the expression of the lasI synthase gene and rsaL, coding for the lasI transcriptional repressor RsaL. Here, single-cell-level gene expression analyses performed in ad hoc engineered biosensor strains and deletion mutants revealed that direct binding of RsaL to the lasI promoter region increases heterogeneous activation of the las QS system. Experiments performed with a dual-fluorescence reporter system showed that the LasR-dependent expression of lasI and rsaL does not correlate in single cells, indicating that RsaL acts as a brake that stochastically limits the transition of non-quorate cells to the quorate state in a subpopulation of cells expressing high levels of this negative regulator. Interestingly, the rhl QS system that is not controlled by an analogous RsaL protein showed higher homogeneity with respect to the las system. IMPORTANCE Single-cell analyses can reveal that despite experiencing identical physico-chemical conditions, individual bacterial cells within a monoclonal population may exhibit variations in gene expression. Such phenotypic heterogeneity has been described for several aspects of bacterial physiology, including QS activation. This study demonstrates that the transition of non-quorate cells to the quorate state is a graded process that does not occur at a specific cell density and that subpopulations of non-quorate cells also persist at high cell density. Here, we provide a mechanistic explanation for this phenomenon, showing that a negative feedback regulatory loop integrated into the las system has a pivotal role in promoting cell-to-cell variation in the QS activation state and in limiting the transition of non-quorate cells to the quorate state in P. aeruginosa.

Alkyl-quinolone-dependent quorum sensing controls prophage-mediated autolysis in Pseudomonas aeruginosa colony biofilms.

Pseudomonas aeruginosa is a model quorum sensing (QS) pathogen with three interconnected QS circuits that control the production of virulence factors and antibiotic tolerant biofilms. The pqs QS system of P. aeruginosa is responsible for the biosynthesis of diverse 2-alkyl-4-quinolones (AQs), of which 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) function as QS signal molecules. Transcriptomic analyses revealed that HHQ and PQS influenced the expression of multiple genes via PqsR-dependent and -independent pathways whereas 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) had no effect on P. aeruginosa transcriptome. HQNO is a cytochrome bc 1 inhibitor that causes P. aeruginosa programmed cell death and autolysis. However, P. aeruginosa pqsL mutants unable to synthesize HQNO undergo autolysis when grown as colony biofilms. The mechanism by which such autolysis occurs is not understood. Through the generation and phenotypic characterization of multiple P. aeruginosa PAO1 mutants producing altered levels of AQs in different combinations, we demonstrate that mutation of pqsL results in the accumulation of HHQ which in turn leads to Pf4 prophage activation and consequently autolysis. Notably, the effect of HHQ on Pf4 activation is not mediated via its cognate receptor PqsR. These data indicate that the synthesis of HQNO in PAO1 limits HHQ-induced autolysis mediated by Pf4 in colony biofilms. A similar phenomenon is shown to occur in P. aeruginosa cystic fibrosis (CF) isolates, in which the autolytic phenotype can be abrogated by ectopic expression of pqsL.

ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa.

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.

Mushroom-shaped structures formed in Acinetobacter baumannii biofilms grown in a roller bioreactor are associated with quorum sensing-dependent Csu-pilus assembly.

There is currently a need to develop simple biofilm models that facilitate investigation of the architecture/biology of mature bacterial biofilms in a consistent/standardized manner given their environmental and clinical importance and the need for new anti-biofilm interventions. This study introduces a novel biofilm culture system termed the rolling biofilm bioreactor (RBB). This easily operated system allows adherent microbial cells to be repeatedly exposed to air/solid/liquid interfaces optimizing biofilm growth. The RBB was exploited to investigate biofilm formation in Acinetobacter baumannii. High levels of A. baumannii biofilm biomass reproducibly accumulate in the RBB and, importantly, undergo a maturation step to form large mushroom-shaped structures that had not been observed in other models. Based on image analysis of biofilm development and genetic manipulation, we show how N-acylhomoserine lactone-dependent quorum sensing (QS) impacts on biofilm differentiation, composition and antibiotic tolerance. Our results indicate that extracellular DNA (eDNA) is a key matrix component in mature Acinetobacter biofilms as the mushroom-like structures consist of dense cellular masses encased in an eDNA mesh. Moreover, this study reveals the contribution of QS to A. baumannii biofilm differentiation through Csu pilus assembly regulation. Understanding the mechanisms of structural development of mature biofilms helps to identify new biofilm eradication and removal strategies.

Actinomadura graeca sp. nov.: A novel producer of the macrocyclic antibiotic zelkovamycin.

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide identity (ANI) value of 89% with A. macra, the highest among related species. Further phenotypic and chemotaxonomic analyses confirmed that the isolate represents a previously uncharacterised species in the genus Actinomadura, for which the name Actinomadura graeca sp. nov. is proposed (type strain 32-07T). The G+C content of A. graeca 32-07 is 72.36%. The cell wall contains DL-diaminopimelic acid, intracellular sugars are glucose, ribose and galactose, the predominant menaquinone is MK-9(H6), the major cellular lipid is phosphatidylinositol and fatty acids consist mainly of hexadecanoic acid. No mycolic acid was detected. Furthermore, A. graeca 32-07 has been confirmed as a novel producer of the non-ribosomal peptide antibiotic zelkovamycin and we report herein a provisional description of the unique biosynthetic gene cluster.

Design and Evaluation of New Quinazolin-4(3H)-one Derived PqsR Antagonists as Quorum Sensing Quenchers in Pseudomonas aeruginosa.

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of antivirulence therapeutics to tackle bacterial infections has attracted considerable attention over the past decades as they hamper the pathogenicity of target microbes with reduced selective pressure, minimizing the emergence of resistance. One such approach is to interfere with the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and host-microbe interactions. In this study, we report the hit identification and optimization of PqsR antagonists using virtual screening coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA PpqsA promoter controlled by PqsR with an IC50 of 1 µM. Using isothermal titration calorimetry, a Kd of 10 nM for the PqsR ligand binding domain (PqsRLBD) was determined for 61. Furthermore, the crystal structure of 61 with PqsRLBD was attained with a resolution of 2.65 Å. Compound 61 significantly reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this compound potentiated the effect of ciprofloxacin in early stages of biofilm treatment and in Galleria mellonella infected with PA. Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identification of a PA QS inhibitor which can be advanced into preclinical development.

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through lack of the Rieske cytochrome bc1 sub-unit.

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of PQS-dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat substrate(s) responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the primary signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. This defect was also phenocopied by deletion of cytB or cytC1. Thus, either lack of the Rieske sub-unit or mutation of cytochrome bc1 genes results in the perturbation of PQS-dependent autoinduction resulting in eDNA deficient biofilms, reduced antibiotic tolerance and compromised virulence factor production.

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol.

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of ß-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO2) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol-1 h-1 autotrophically. This is first report of (R)-1,3-BDO production from CO2.

Combining Inducible Lectin Expression and Magnetic Glyconanoparticles for the Selective Isolation of Bacteria from Mixed Populations.

The selective isolation of bacteria from mixed populations has been investigated in varied applications ranging from differential pathogen identification in medical diagnostics and food safety to the monitoring of microbial stress dynamics in industrial bioreactors. Selective isolation techniques are generally limited to the confinement of small populations in defined locations, may be unable to target specific bacteria, or rely on immunomagnetic separation, which is not universally applicable. In this proof-of-concept work, we describe a novel strategy combining inducible bacterial lectin expression with magnetic glyconanoparticles (MGNPs) as a platform technology to enable selective bacterial isolation from cocultures. An inducible mutant of the type 1 fimbriae, displaying the mannose-specific lectin FimH, was constructed in Escherichia coli allowing for "on-demand" glycan-binding protein presentation following external chemical stimulation. Binding to glycopolymers was only observed upon fimbrial induction and was specific for mannosylated materials. A library of MGNPs was produced via the grafting of well-defined catechol-terminal glycopolymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization to magnetic nanoparticles. Thermal analysis revealed high functionalization (≥85% polymer by weight). Delivery of MGNPs to cocultures of fluorescently labeled bacteria followed by magnetic extraction resulted in efficient depletion of type 1 fimbriated target cells from wild-type or afimbriate E. coli. Extraction efficiency was found to be dependent on the molecular weight of the glycopolymers utilized to engineer the nanoparticles, with MGNPs decorated with shorter Dopa-(ManAA)50 mannosylated glycopolymers found to perform better than those assembled from a longer Dopa-(ManAA)200 analogue. The extraction efficiency of fimbriated E. coli was also improved when the counterpart strain did not harbor the genetic apparatus for the expression of the type 1 fimbriae. Overall, this work suggests that the modulation of the genetic apparatus encoding bacterial surface-associated lectins coupled with capture through MGNPs could be a versatile tool for the extraction of bacteria from mixed populations.

Genome-Wide Analysis of Targets for Post-Transcriptional Regulation by Rsm Proteins in Pseudomonas putida.

Post-transcriptional regulation is an important step in the control of bacterial gene expression in response to environmental and cellular signals. Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences bound in vivo by each of these proteins. Affinity purification and sequencing of RNA molecules associated with Rsm proteins were used to discover direct binding targets, corresponding to 437 unique RNA molecules, 75 of them being common to the three proteins. Relevant targets include genes encoding proteins involved in signal transduction and regulation, metabolism, transport and secretion, stress responses, and the turnover of the intracellular second messenger c-di-GMP. To our knowledge, this is the first combined global analysis in a bacterium harboring three Rsm homologs. It offers a broad overview of the network of processes subjected to this type of regulation and opens the way to define what are the sequence and structure determinants that define common or differential recognition of specific RNA molecules by these proteins.

Hit Identification of New Potent PqsR Antagonists as Inhibitors of Quorum Sensing in Planktonic and Biofilm Grown Pseudomonas aeruginosa.

Current treatments for Pseudomonas aeruginosa infections are becoming less effective because of the increasing rates of multi-antibiotic resistance. Pharmacological targeting of virulence through inhibition of quorum sensing (QS) dependent virulence gene regulation has considerable therapeutic potential. In P. aeruginosa, the pqs QS system regulates the production of multiple virulence factors as well as biofilm maturation and is a promising approach for developing antimicrobial adjuvants for combatting drug resistance. In this work, we report the hit optimisation for a series of potent novel inhibitors of PqsR, a key regulator of the pqs system, bearing a 2-((5-methyl-5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio) acetamide scaffold. The initial hit compound 7 (PAO1-L IC50 0.98 ± 0.02 µM, PA14 inactive at 10 µM) was obtained through a virtual screening campaign performed on the PqsR ligand binding domain using the University of Nottingham Managed Chemical Compound Collection. Hit optimisation gave compounds with enhanced potency against strains PAO1-L and PA14, evaluated using P. aeruginosa pqs-based QS bioreporter assays. Compound 40 (PAO1-L IC50 0.25 ± 0.12 µM, PA14 IC50 0.34 ± 0.03 µM) is one of the most potent PqsR antagonists reported showing significant inhibition of P. aeruginosa pyocyanin production and pqs system signaling in both planktonic cultures and biofilms. The co-crystal structure of 40 with the PqsR ligand binding domain revealed the specific binding interactions occurring between inhibitor and this key regulatory protein.

Differential Regulation of the Phenazine Biosynthetic Operons by Quorum Sensing in Pseudomonas aeruginosa PAO1-N.

The Pseudomonas aeruginosa quorum sensing (QS) network plays a key role in the adaptation to environmental changes and the control of virulence factor production in this opportunistic human pathogen. Three interlinked QS systems, namely las, rhl, and pqs, are central to the production of pyocyanin, a phenazine virulence factor which is typically used as phenotypic marker for analysing QS. Pyocyanin production in P. aeruginosa is a complex process involving two almost identical operons termed phzA1B1C1D1E1F1G1 (phz1) and phzA2B2C2D2E2F2G2 (phz2), which drive the production of phenazine-1-carboxylic acid (PCA) which is further converted to pyocyanin by two modifying enzymes PhzM and PhzS. Due to the high sequence conservation between the phz1 and phz2 operons (nucleotide identity > 98%), analysis of their individual expression by RNA hybridization, qRT-PCR or transcriptomics is challenging. To overcome this difficulty, we utilized luminescence based promoter fusions of each phenazine operon to measure in planktonic cultures their transcriptional activity in P. aeruginosa PAO1-N genetic backgrounds impaired in different components of the las, rhl, and pqs QS systems, in the presence or absence of different QS signal molecules. Using this approach, we found that all three QS systems play a role in differentially regulating the phz1 and phz2 phenazine operons, thus uncovering a higher level of complexity to the QS regulation of PCA biosynthesis in P. aeruginosa than previously appreciated. Importance: The way the P. aeruginosa QS regulatory networks are intertwined creates a challenge when analysing the mechanisms governing specific QS-regulated traits. Multiple QS regulators and signals have been associated with the production of phenazine virulence factors. In this work we designed experiments where we dissected the contribution of specific QS switches using individual mutations and complementation strategies to gain further understanding of the specific roles of these QS elements in controlling expression of the two P. aeruginosa phenazine operons. Using this approach we have teased out which QS regulators have either indirect or direct effects on the regulation of the two phenazine biosynthetic operons. The data obtained highlight the sophistication of the QS cascade in P. aeruginosa and the challenges in analysing the control of phenazine secondary metabolites.

Genome-wide mapping of the RNA targets of the Pseudomonas aeruginosa riboregulatory protein RsmN.

Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In Pseudomonas aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities of RsmN for its target RNAs and overall impact on the biology of this pathogen. We purified and mapped 503 transcripts directly bound by RsmN in P. aeruginosa. About 200 of the mRNAs identified encode proteins of demonstrated function including some determining acute and chronic virulence traits. For example, RsmN reduces biofilm development both directly and indirectly via multiple pathways, involving control of Pel exopolysaccharide biosynthesis and c-di-GMP levels. The RsmN targets identified are also shared with RsmA, although deletion of rsmN generally results in less pronounced phenotypes than those observed for ΔrsmA or ΔrsmArsmNind mutants, probably as a consequence of different binding affinities. Targets newly identified for the Rsm system include the small non-coding RNA CrcZ involved in carbon catabolite repression, for which differential binding of RsmN and RsmA to specific CrcZ regions is demonstrated. The results presented here provide new insights into the intricacy of riboregulatory networks involving multiple but distinct RsmA homologues.

2-Tridecanone impacts surface-associated bacterial behaviours and hinders plant-bacteria interactions.

Surface motility and biofilm formation are behaviours which enable bacteria to infect their hosts and are controlled by different chemical signals. In the plant symbiotic alpha-proteobacterium Sinorhizobium meliloti, the lack of long-chain fatty acyl-coenzyme A synthetase activity (FadD) leads to increased surface motility, defects in biofilm development and impaired root colonization. In this study, analyses of lipid extracts and volatiles revealed that a fadD mutant accumulates 2-tridecanone (2-TDC), a methylketone (MK) known as a natural insecticide. Application of pure 2-TDC to the wild-type strain phenocopies the free-living and symbiotic behaviours of the fadD mutant. Structural features of the MK determine its ability to promote S. meliloti surface translocation, which is mainly mediated by a flagella-independent motility. Transcriptomic analyses showed that 2-TDC induces differential expression of iron uptake, redox and stress-related genes. Interestingly, this MK also influences surface motility and impairs biofilm formation in plant and animal pathogenic bacteria. Moreover, 2-TDC not only hampers alfalfa nodulation but also the development of tomato bacterial speck disease. This work assigns a new role to 2-TDC as an infochemical that affects important bacterial traits and hampers plant-bacteria interactions by interfering with microbial colonization of plant tissues.

Functional identification of the prnABCD operon and its regulation in Serratia plymuthica.

The antibiotic pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite that plays an important role in the biocontrol of plant diseases due to its broad-spectrum of antimicrobial activities. The PRN biosynthetic gene cluster remains to be characterised in Serratia plymuthica, though it is highly conserved in PRN-producing bacteria. To better understand PRN biosynthesis and its regulation in Serratia, the prnABCD operon from S. plymuthica G3 was cloned, sequenced and expressed in Escherichia coli DH5α. Furthermore, an engineered strain prnind which is a conditional mutant of G3 prnABCD under the control of the Ptac promoter was constructed. This mutant was able to overproduce PRN with isopropylthiogalactoside (IPTG) induction by overexpressing prnABCD, whilst behaving as a conditional mutant of G3 prnABCD in the absence of IPTG. These results confirmed that prnABCD is responsible for PRN biosynthesis in strain G3. Further experiments involving lux-/dsRed-based promoter fusions, combined with site-directed mutagenesis of the putative σS extended -10 region in the prnA promoter, and liquid chromatography-mass spectrometry (LC-MS) analysis extended our previous knowledge about G3, revealing that quorum sensing (QS) regulates PRN biosynthesis through cross talk with RpoS, which may directly activated prnABCD transcription. These findings suggest that PRN in S. plymuthica G3 is produced in a tightly controlled manner, and has diverse functions, such as modulation of cell motility, in addition to antimicrobial activities. Meanwhile, the construction of inducible mutants could be a powerful tool to improve PRN production, beyond its potential use for the investigation of the biological function of PRN.

In Silico and in Vitro-Guided Identification of Inhibitors of Alkylquinolone-Dependent Quorum Sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections, posing a serious burden to public health, due to its antibiotic resistance. The P. aeruginosa Pseudomonas Quinolone System (pqs) quorum sensing system, driven by the activation of the transcriptional regulator, PqsR (MvfR) by alkylquinolone (AQ) signal molecules, is a key player in the regulation of virulence and a potential target for the development of novel antibacterial agents. In this study, we performed in silico docking analysis, coupled with screening using a P. aeruginosa mCTX::PpqsA-lux chromosomal promoter fusion, to identify a series of new PqsR antagonists. The hit compounds inhibited pyocyanin and alkylquinolone signal molecule production in P. aeruginosa PAO1-L and PA14 strains. The inhibitor Ia, which showed the highest activity in PA14, reduced biofilm formation in PAO1-L and PA14, increasing their sensitivity to tobramycin. Furthermore, the hepatic and plasma stabilities for these compounds were determined in both rat and human in vitro microsomal assays, to gain a further understanding of their therapeutic potential. This work has uncovered a new class of P. aeruginosa PqsR antagonists with potential for hit to lead optimisation in the search for quorum sensing inhibitors for future anti-infective drug discovery programs.