Exploring the synthetic potential of a marine transaminase including discrimination at a remote stereocentre.

The marine transaminase, P-ω-TA, can be employed for the transamination from 1-aminotetralins and 1-aminoindanes with differentiation of stereochemistry at both the site of reaction and at a remote stereocentre resulting in formation of ketone products with up to 93% ee. While 4-substituents are tolerated on the tetralin core, the presence of 3- or 8-substituents is not tolerated by the transaminase. In general P-ω-TA shows capacity for remote diastereoselectivity, although both the stereoselectivity and efficiency are dependent on the specific substrate structure. Optimum efficiency and selectivity are seen with 4-haloaryl-1-aminotetralins and 3-haloaryl-1-aminoindanes, which may be associated with the marine origin of this enzyme.

A structure-function analysis of interspecies antagonism by the 2-heptyl-4-alkyl-quinolone signal molecule from Pseudomonas aeruginosa.

In recent years, the alkyl-quinolone molecular framework has already provided a rich source of bioactivity for the development of novel anti-infective compounds. Based on the quorum-sensing signalling molecules 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS) from the nosocomial pathogen Pseudomonas aeruginosa, modifications have been developed with markedly enhanced anti-biofilm bioactivity towards important fungal and bacterial pathogens, including Candida albicans and Aspergillus fumigatus. Here we show that antibacterial activity of HHQ against Vibrionaceae is species-specific and it requires an exquisite level of structural fidelity within the alkyl-quinolone molecular framework. Antibacterial activity was demonstrated against the serious human pathogens Vibrio vulnificus and Vibrio cholerae as well as a panel of bioluminescent squid symbiont Allivibrio fischeri isolates. In contrast, Vibrio parahaemolyticus growth and biofilm formation was unaffected in the presence of HHQ and all the structural variants tested. In general, modification to almost all of the molecule except the alkyl-chain end, led to loss of activity. This suggests that the bacteriostatic activity of HHQ requires the concerted action of the entire framework components. The only exception to this pattern was deuteration of HHQ at the C3 position. HHQ modified with a terminal alkene at the quinolone alkyl chain retained bacteriostatic activity and was also found to activate PqsR signalling comparable to the native agonist. The data from this integrated analysis provides novel insights into the structural flexibility underpinning the signalling activity of the complex alkyl-quinolone molecular communication system.

Analogues of Pseudomonas aeruginosa signalling molecules to tackle infections.

The emergence of antibiotic resistance coupled with the lack of investment by pharmaceutical companies necessitates a new look at how we tackle bacterial infections. An intriguing tactic is the interruption of bacterial communication systems. This non-biocidal approach would circumvent the evolutionary pressure on bacteria to mutate and develop resistance. In many pathogenic microorganisms, communication systems, collectively termed quorum sensing (QS), have been observed to control a number of bacterial behaviours including expression of virulence factors and the development of biofilms. QS signalling molecules and their biomimetics, therefore, represent a rational target for the disruption of cooperative behaviour and thus the development of novel antimicrobial strategies. Herein we review recent developments towards the interference of Pseudomonas aeruginosa QS using signalling molecules and their mimetics.

Coumarin: a novel player in microbial quorum sensing and biofilm formation inhibition.

Antibiotic resistance is a growing threat worldwide, causing serious problems in the treatment of microbial infections. The discovery and development of new drugs is urgently needed to overcome this problem which has greatly undermined the clinical effectiveness of conventional antibiotics. An intricate cell-cell communication system termed quorum sensing (QS) and the coordinated multicellular behaviour of biofilm formation have both been identified as promising targets for the treatment and clinical management of microbial infections. QS systems allow bacteria to adapt rapidly to harsh conditions, and are known to promote the formation of antibiotic tolerant biofilm communities. It is well known that biofilm is a recalcitrant mode of growth and it also increases bacterial resistance to conventional antibiotics. The pharmacological properties of coumarins have been well described, and these have included several that possess antimicrobial properties. More recently, reports have highlighted the potential role of coumarins as alternative therapeutic strategies based on their ability to block the QS signalling systems and to inhibit the formation of biofilms in clinically relevant pathogens. In addition to human infections, coumarins have also been found to be effective in controlling plant pathogens, infections in aquaculture, food spoilage and in reducing biofouling caused by eukaryotic organisms. Thus, the coumarin class of small molecule natural product are emerging as a promising strategy to combat bacterial infections in the new era of antimicrobial resistance.

Harnessing Marine Biocatalytic Reservoirs for Green Chemistry Applications through Metagenomic Technologies.

In a demanding commercial world, large-scale chemical processes have been widely utilised to satisfy consumer related needs. Chemical industries are key to promoting economic growth and meeting the requirements of a sustainable industrialised society. The market need for diverse commodities produced by the chemical industry is rapidly expanding globally. Accompanying this demand is an increased threat to the environment and to human health, due to waste produced by increased industrial production. This increased demand has underscored the necessity to increase reaction efficiencies, in order to reduce costs and increase profits. The discovery of novel biocatalysts is a key method aimed at combating these difficulties. Metagenomic technology, as a tool for uncovering novel biocatalysts, has great potential and applicability and has already delivered many successful achievements. In this review we discuss, recent developments and achievements in the field of biocatalysis. We highlight how green chemistry principles through the application of biocatalysis, can be successfully promoted and implemented in various industrial sectors. In addition, we demonstrate how two novel lipases/esterases were mined from the marine environment by metagenomic analysis. Collectively these improvements can result in increased efficiency, decreased energy consumption, reduced waste and cost savings for the chemical industry.

The requirements at the C-3 position of alkylquinolones for signalling in Pseudomonas aeruginosa.

The 'perfect storm' of increasing bacterial antibiotic resistance and a decline in the discovery of new antibiotics, has made it necessary to search for new and innovative strategies to treat bacterial infections. Interruption of bacterial cell-to-cell communication signalling (Quorum Sensing), thus neutralizing virulence in pathogenic bacteria, is a growing area. 2-Alkyl-4-quinolones, HHQ and PQS, play a key role in the quorum sensing circuitry of P. aeruginosa. We report a new set of isosteres of 2-heptyl-6-nitroquinolin-4-one, with alterations at C-3, and evaluate the key structural requirements for agonistic and antagonistic activity in Pseudomonas aeruginosa.

Is There Potential for Repurposing Statins as Novel Antimicrobials?

Statins are members of a class of pharmaceutical widely used to reduce high levels of serum cholesterol. In addition, statins have so-called "pleiotropic effects," which include inflammation reduction, immunomodulation, and antimicrobial effects. An increasing number of studies are emerging which detail the attenuation of bacterial growth and in vitro and in vivo virulence by statin treatment. In this review, we describe the current information available concerning the effects of statins on bacterial infections and provide insight regarding the potential use of these compounds as antimicrobial therapeutic agents.

Exploiting Interkingdom Interactions for Development of Small-Molecule Inhibitors of Candida albicans Biofilm Formation.

A rapid decline in the development of new antimicrobial therapeutics has coincided with the emergence of new and more aggressive multidrug-resistant pathogens. Pathogens are protected from antibiotic activity by their ability to enter an aggregative biofilm state. Therefore, disrupting this process in pathogens is a key strategy for the development of next-generation antimicrobials. Here, we present a suite of compounds, based on the Pseudomonas aeruginosa 2-heptyl-4(1H)-quinolone (HHQ) core quinolone interkingdom signal structure, that exhibit noncytotoxic antibiofilm activity toward the fungal pathogen Candida albicans In addition to providing new insights into what is a clinically important bacterium-fungus interaction, the capacity to modularize the functionality of the quinolone signals is an important advance in harnessing the therapeutic potential of signaling molecules in general. This provides a platform for the development of potent next-generation small-molecule therapeutics targeting clinically relevant fungal pathogens.

Dissecting the regulation of bile-induced biofilm formation in Staphylococcus aureus.

Aspiration of bile into the cystic fibrosis (CF) lung has emerged as a prognostic factor for reduced microbial lung biodiversity and the establishment of often fatal, chronic pathogen infections. Staphylococcus aureus is one of the earliest pathogens detected in the lungs of children with CF, and once established as a chronic infection, strategies for its eradication become limited. Several lung pathogens are stimulated to produce biofilms in vitro in the presence of bile. In this study, we further investigated the effects of bile on S. aureus biofilm formation. Most clinical S. aureus strains and the laboratory strain RN4220 were stimulated to form biofilms with sub-inhibitory concentrations of bovine bile. Additionally, we observed bile-induced sensitivity to aminoglycosides, which we exploited in a bursa aurealis transposon screen to isolate mutants reduced in aminoglycoside sensitivity and augmented in bile-induced biofilm formation. We identified five mutants that exhibited hypersensitivity to bile with respect to bile-induced biofilm formation, three of which carried transposon insertions within gene clusters involved in wall teichoic acid (WTA) biosynthesis or transport. Strain TM4 carried an insertion between the divergently oriented tagH and tagG genes, which encode the putative WTA membrane translocation apparatus. Ectopic expression of tagG in TM4 restored a wild-type bile-induced biofilm response, suggesting that reduced translocation of WTA in TM4 induced sensitivity to bile and enhanced the bile-induced biofilm formation response. We propose that WTA may be important for protecting S. aureus against exposure to bile and that bile-induced biofilm formation may be an evolved response to protect cells from bile-induced cell lysis.

Synthesis and electrochemical detection of a thiazolyl-indole natural product isolated from the nosocomial pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen, capable of surviving in a broad range of natural environments and quickly acquiring resistance. It is associated with hospital-acquired infections, particularly in patients with compromised immunity, and is the primary cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa is also of nosocomial importance on dairy farms and veterinary hospitals, where it is a key morbidity factor in bovine mastitis. P. aeruginosa uses a cell-cell communication system consisting of signalling molecules to coordinate bacterial secondary metabolites, biofilm formation, and virulence. Simple and sensitive methods for the detection of biomolecules as indicators of P. aeruginosa infection would be of great clinical importance. Here, we report the synthesis of the P. aeruginosa natural product, barakacin, which was recently isolated from the bovine ruminal strain ZIO. A simple and sensitive electrochemical method was used for barakacin detection using a boron-doped diamond (BDD) and glassy carbon (GC) electrodes, based on cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The influence of electrolyte pH on the peak potential and peak currents was also investigated. At pH 2.0, the peak current was linearly dependent on barakacin concentration (in the range used, 1-10 µM), with correlation coefficients greater than 0.98 on both electrodes. The detection limit (S/N = 3) on the BDD electrode was 100-fold lower than that obtained on the GC electrode. The optimized method using the BDD electrode was extended to bovine (cow feces) and human (sputum of a CF patient) samples. Spiked barakacin was easily detected in these matrices at a limit of 0.5 and 0.05 µM, respectively. Graphical abstract Electrochemical detection of barakacin.

Integrated (Meta) Genomic and Synthetic Biology Approaches to Develop New Biocatalysts.

In recent years, the marine environment has been the subject of increasing attention from biotechnological and pharmaceutical industries as a valuable and promising source of novel bioactive compounds. Marine biodiscovery programmes have begun to reveal the extent of novel compounds encoded within the enormous bacterial richness and diversity of the marine ecosystem. A combination of unique physicochemical properties and spatial niche-specific substrates, in wide-ranging and extreme habitats, underscores the potential of the marine environment to deliver on functionally novel biocatalytic activities. With the growing need for green alternatives to industrial processes, and the unique transformations which nature is capable of performing, marine biocatalysts have the potential to markedly improve current industrial pipelines. Furthermore, biocatalysts are known to possess chiral selectivity and specificity, a key focus of pharmaceutical drug design. In this review, we discuss how the explosion in genomics based sequence analysis, allied with parallel developments in synthetic and molecular biology, have the potential to fast-track the discovery and subsequent improvement of a new generation of marine biocatalysts.

A structure activity-relationship study of the bacterial signal molecule HHQ reveals swarming motility inhibition in Bacillus atrophaeus.

The sharp rise in antimicrobial resistance has been matched by a decline in the identification and clinical introduction of new classes of drugs to target microbial infections. Thus new approaches are being sought to counter the pending threat of a post-antibiotic era. In that context, the use of non-growth limiting small molecules, that target virulence behaviour in pathogens, has emerged as a solution with real clinical potential. We have previously shown that two signal molecules (HHQ and PQS) from the nosocomial pathogen Pseudomonas aeruginosa have modulatory activity towards other microorganisms. This current study involves the synthesis and evaluation of analogues of HHQ towards swarming and biofilm virulence behaviour in Bacillus atrophaeus, a soil bacterium and co-inhibitor with P. aeruginosa. Compounds with altered C6-C8 positions on the anthranilate-derived ring of HHQ, display a surprising degree of biological specificity, with certain candidates displaying complete motility inhibition. In contrast, anti-biofilm activity of the parent molecule was completely lost upon alteration at any position indicating a remarkable degree of specificity and delineation of phenotype.

Deciphering the role of coumarin as a novel quorum sensing inhibitor suppressing virulence phenotypes in bacterial pathogens.

The rapid unchecked rise in antibiotic resistance over the last few decades has led to an increased focus on the need for alternative therapeutic strategies for the treatment and clinical management of microbial infections. In particular, small molecules that can suppress microbial virulence systems independent of any impact on growth are receiving increased attention. Quorum sensing (QS) is a cell-to-cell signalling communication system that controls the virulence behaviour of a broad spectrum of bacterial pathogens. QS systems have been proposed as an effective target, particularly as they control biofilm formation in pathogens, a key driver of antibiotic ineffectiveness. In this study, we identified coumarin, a natural plant phenolic compound, as a novel QS inhibitor, with potent anti-virulence activity in a broad spectrum of pathogens. Using a range of biosensor systems, coumarin was active against short, medium and long chain N-acyl-homoserine lactones, independent of any effect on growth. To determine if this suppression was linked to anti-virulence activity, key virulence systems were studied in the nosocomial pathogen Pseudomonas aeruginosa. Consistent with suppression of QS, coumarin inhibited biofilm, the production of phenazines and swarming motility in this organism potentially linked to reduced expression of the rhlI and pqsA quorum sensing genes. Furthermore, coumarin significantly inhibited biofilm formation and protease activity in other bacterial pathogens and inhibited bioluminescence in Aliivibrio fischeri. In light of these findings, coumarin would appear to have potential as a novel quorum sensing inhibitor with a broad spectrum of action.

The Sound of Silence: Activating Silent Biosynthetic Gene Clusters in Marine Microorganisms.

Unlocking the rich harvest of marine microbial ecosystems has the potential to both safeguard the existence of our species for the future, while also presenting significant lifestyle benefits for commercial gain. However, while significant advances have been made in the field of marine biodiscovery, leading to the introduction of new classes of therapeutics for clinical medicine, cosmetics and industrial products, much of what this natural ecosystem has to offer is locked in, and essentially hidden from our screening methods. Releasing this silent potential represents a significant technological challenge, the key to which is a comprehensive understanding of what controls these systems. Heterologous expression systems have been successful in awakening a number of these cryptic marine biosynthetic gene clusters (BGCs). However, this approach is limited by the typically large size of the encoding sequences. More recently, focus has shifted to the regulatory proteins associated with each BGC, many of which are signal responsive raising the possibility of exogenous activation. Abundant among these are the LysR-type family of transcriptional regulators, which are known to control production of microbial aromatic systems. Although the environmental signals that activate these regulatory systems remain unknown, it offers the exciting possibility of evoking mimic molecules and synthetic expression systems to drive production of potentially novel natural products in microorganisms. Success in this field has the potential to provide a quantum leap forward in medical and industrial bio-product development. To achieve these new endpoints, it is clear that the integrated efforts of bioinformaticians and natural product chemists will be required as we strive to uncover new and potentially unique structures from silent or cryptic marine gene clusters.

Emerging concepts promising new horizons for marine biodiscovery and synthetic biology.

The vast oceans of the world, which comprise a huge variety of unique ecosystems, are emerging as a rich and relatively untapped source of novel bioactive compounds with invaluable biotechnological and pharmaceutical potential. Evidence accumulated over the last decade has revealed that the diversity of marine microorganisms is enormous with many thousands of bacterial species detected that were previously unknown. Associated with this diversity is the production of diverse repertoires of bioactive compounds ranging from peptides and enzymes to more complex secondary metabolites that have significant bioactivity and thus the potential to be exploited for innovative biotechnology. Here we review the discovery and functional potential of marine bioactive peptides such as lantibiotics, nanoantibiotics and peptidomimetics, which have received particular attention in recent years in light of their broad spectrum of bioactivity. The significance of marine peptides in cell-to-cell communication and how this may be exploited in the discovery of novel bioactivity is also explored. Finally, with the recent advances in bioinformatics and synthetic biology, it is becoming clear that the integration of these disciplines with genetic and biochemical characterization of the novel marine peptides, offers the most potential in the development of the next generation of societal solutions.

Bile acids repress hypoxia-inducible factor 1 signaling and modulate the airway immune response.

Gastroesophageal reflux (GER) frequently occurs in patients with respiratory disease and is particularly prevalent in patients with cystic fibrosis. GER is a condition in which the duodenogastric contents of the stomach leak into the esophagus, in many cases resulting in aspiration into the respiratory tract. As such, the presence of GER-derived bile acids (BAs) has been confirmed in the bronchoalveolar lavage fluid and sputum of affected patients. We have recently shown that bile causes cystic fibrosis-associated bacterial pathogens to adopt a chronic lifestyle and may constitute a major host trigger underlying respiratory infection. The current study shows that BAs elicit a specific response in humans in which they repress hypoxia-inducible factor 1α (HIF-1α) protein, an emerging master regulator in response to infection and inflammation. HIF-1α repression was shown to occur through the 26S proteasome machinery via the prolyl hydroxylase domain (PHD) pathway. Further analysis of the downstream inflammatory response showed that HIF-1α repression by BAs can significantly modulate the immune response of airway epithelial cells, correlating with a decrease in interleukin-8 (IL-8) production, while IL-6 production was strongly increased. Importantly, the effects of BAs on cytokine production can also be more dominant than the bacterium-mediated effects. However, the effect of BAs on cytokine levels cannot be fully explained by their ability to repress HIF-1α, which is not surprising, given the complexity of the immune regulatory network. The suppression of HIF-1 signaling by bile acids may have a significant influence on the progression and outcome of respiratory disease, and the molecular mechanism underpinning this response warrants further investigation.

Inhibition of co-colonizing cystic fibrosis-associated pathogens by Pseudomonas aeruginosa and Burkholderia multivorans.

Cystic fibrosis (CF) is a recessive genetic disease characterized by chronic respiratory infections and inflammation causing permanent lung damage. Recurrent infections are caused by Gram-negative antibiotic-resistant bacterial pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen genus Pandoraea. In this study, the interactions between co-colonizing CF pathogens were investigated. Both Pandoraea and Bcc elicited potent pro-inflammatory responses that were significantly greater than Ps. aeruginosa. The original aim was to examine whether combinations of pro-inflammatory pathogens would further exacerbate inflammation. In contrast, when these pathogens were colonized in the presence of Ps. aeruginosa the pro-inflammatory response was significantly decreased. Real-time PCR quantification of bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the growth of Burkholderia multivorans, Burkholderia cenocepacia, Pandoraea pulmonicola and Pandoraea apista, which may be a factor in its dominance as a colonizer of CF patients. Ps. aeruginosa cell-free supernatant also suppressed growth of these pathogens, indicating that inhibition was innate rather than a response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia strain J2315. B. multivorans was the only species that could significantly inhibit Ps. aeruginosa growth. B. multivorans also inhibited B. cenocepacia and Pa. apista. In conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and virulence of co-colonizing CF pathogens.

A new regulator of pathogenicity (bvlR) is required for full virulence and tight microcolony formation in Pseudomonas aeruginosa.

LysR-type transcriptional regulators (LTTRs) are the most common family of transcriptional regulators found in the opportunistic pathogen Pseudomonas aeruginosa. They are known to regulate a wide variety of virulence determinants and have emerged recently as positive global regulators of pathogenicity in a broad spectrum of important bacterial pathogens. However, in spite of their key role in modulating expression of key virulence determinants underpinning pathogenic traits associated with the process of infection, surprisingly few are found to be transcriptionally altered by contact with host cells. BvlR (PA14_26880) an LTTR of previously unknown function, has been shown to be induced in response to host cell contact, and was therefore investigated for its potential role in virulence. BvlR expression was found to play a pivotal role in the regulation of acute virulence determinants such as type III secretion system and exotoxin A production. BvlR also played a key role in P. aeruginosa pathogenicity within the Caenorhabditis elegans acute model of infection. Loss of BvlR led to an inability to form tight microcolonies, a key step in biofilm formation in the cystic fibrosis lung, although surface attachment was increased. Unusually for LTTRs, BvlR was shown to exert its influence through the transcriptional repression of many genes, including the virulence-associated cupA and alg genes. This highlights the importance of BvlR as a new virulence regulator in P. aeruginosa with a central role in modulating key events in the pathogen-host interactome.

Characterisation of non-autoinducing tropodithietic Acid (TDA) production from marine sponge Pseudovibrio species.

The search for new antimicrobial compounds has gained added momentum in recent years, paralleled by the exponential rise in resistance to most known classes of current antibiotics. While modifications of existing drugs have brought some limited clinical success, there remains a critical need for new classes of antimicrobial compound to which key clinical pathogens will be naive. This has provided the context and impetus to marine biodiscovery programmes that seek to isolate and characterize new activities from the aquatic ecosystem. One new antibiotic to emerge from these initiatives is the antibacterial compound tropodithietic acid (TDA). The aim of this study was to provide insight into the bioactivity of and the factors governing the production of TDA in marine Pseudovibrio isolates from a collection of marine sponges. The TDA produced by these Pseudovibrio isolates exhibited potent antimicrobial activity against a broad spectrum of clinical pathogens, while TDA tolerance was frequent in non-TDA producing marine isolates. Comparative genomics analysis suggested a high degree of conservation among the tda biosynthetic clusters while expression studies revealed coordinated regulation of TDA synthesis upon transition from log to stationary phase growth, which was not induced by TDA itself or by the presence of the C10-acyl homoserine lactone quorum sensing signal molecule.