Homeostatic Interplay between Bacterial Cell-Cell Signaling and Iron in Virulence

Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS) networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs) MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL) RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens

Inhibitors of Pathogen Intercellular Signals as Selective Anti-Infective Compounds

Long-term antibiotic use generates pan-resistant super pathogens. Anti-infective compounds that selectively disrupt virulence pathways without affecting cell viability may be used to efficiently combat infections caused by these pathogens. A candidate target pathway is quorum sensing (QS), which many bacterial pathogens use to coordinately regulate virulence determinants. The Pseudomonas aeruginosa MvfR-dependent QS regulatory pathway controls the expression of key virulence genes; and is activated via the extracellular signals 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), whose syntheses depend on anthranilic acid (AA), the primary precursor of 4-hydroxy-2-alkylquinolines (HAQs). Here, we identified halogenated AA analogs that specifically inhibited HAQ biosynthesis and disrupted MvfR-dependent gene expression. These compounds restricted P. aeruginosa systemic dissemination and mortality in mice, without perturbing bacterial viability, and inhibited osmoprotection, a widespread bacterial function. These compounds provide a starting point for the design and development of selective anti-infectives that restrict human P. aeruginosa pathogenesis, and possibly other clinically significant pathogens

La pompe à efflux MexEF-OprN de pseudomonas aeruginosa exporte la molécule de communication intercellulaires 4-hydroxy-2-heptyliquinoline (HHQ)

La bactérie pathogène opportuniste Pseudomonas aeruginosa est la cause d’infections chroniques chez les personnes atteintes de la fibrose kystique (FK). De plus, P. aeruginosa est naturellement très résistante aux antimicrobiens, ce qui rend l’antibiothérapie souvent inefficace. Les pompes à efflux sont des déterminants majeurs de la résistance aux antibiotiques chez cette bactérie. Elles permettent la résistance simultanée à différentes classes d'antibiotiques. La pompe à efflux MexEF-OprN promeut la résistance au chloramphénicol, au triméthoprime, au triclosan et aux fluoroquinolones. L’expression continue de l’opéron mexEF-oprN est observée chez des mutants de mexS, gène dont la fonction est inconnue. Une mutation dans mexS affecte aussi les mécanismes de quorum sensing (QS), c’est-à-dire de communication intercellulaire. Le QS contrôle l’expression de facteurs de virulence et la formation de biofilms, observées dans les voies respiratoires des personnes atteintes de FK. Le QS est contrôlé par des régulateurs transcriptionnels qui, pour être actifs, doivent être couplés à de petites molécules signales nommées auto-inducteurs. Alors que la population bactérienne se densifie, les auto-inducteurs s’accumulent dans le milieu extracellulaire jusqu’à atteindre une concentration-seuil, à laquelle le "quorum" est atteint. MvfR est un régulateur du QS et contrôle l’expression de plusieurs gènes importants pour la virulence, dont ceux de l’opéron pqsABCDE, impliqués dans la biosynthèse des 4-hydroxy-2-alkylquinolines (HAQ). Un auto-inducteur associé à MvfR est le 3,4-dihydroxy-2- heptylquinoline (PQS; Pseudomonas Quinolone Signal). L’enzyme PqsH est responsable de la synthèse du PQS et utilise un HAQ, le 4-hydroxy-2-heptylquinoline (HHQ), comme substrat. Étrangement, la quantité de PQS produit par les mutants mexS est substantiellement réduite. Afin de mieux comprendre l’influence de cette mutation sur les HAQ, nous avons quantifié leurs concentrations par LC-MS/MS chez la souche parentale PA14 et son mutant isogénique mexS. Cette expérience fut effectuée en absence ou présence d’inhibiteurs des pompes à efflux. De plus, le niveau d’expression de plusieurs gènes du QS fût déterminé grâce à des fusions transcriptionnelles entre les régions promotrices des gènes à l’étude et le gène rapporteur lacZ. Mis ensemble, nos résultats démontrent que c’est l’exportation du HHQ par MexEF-OprN qui cause le déficit en PQS. Puisque des souches qui surexpriment mexEF-oprN sont défectueuses pour le QS et moins virulentes, il serait intéressant d’identifier de nouvelles molécules à potentiel thérapeutique qui permettrait de moduler l’activité de la pompe MexEF-OprN. Cela pourrait faciliter le traitement des infections pulmonaires à P. aeruginosa

Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa

<p>Abstract</p> <p>Background</p> <p>The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment that was obtained with a 1- or 3-step PCR protocol and that carries an antibiotic cassette flanked by a region homologous to the target locus into a strain that expresses the lambda Red recombination system.</p> <p>Results</p> <p>This system has been modified for use in <it>Pseudomonas aeruginosa</it>. Chromosomal DNA deletions of single genes were generated using 3-step PCR products containing flanking regions 400–600 nucleotides (nt) in length that are homologous to the target sequence. A 1-step PCR product with a homologous extension flanking region of only 100 nt was in some cases sufficient to obtain the desired mutant. We further showed that the <it>P. aeruginosa </it>strain PA14 non-redundant transposon library can be used in conjunction with the lambda Red technique to rapidly generate large chromosomal deletions or transfer mutated genes into various PA14 isogenic mutants to create multi-locus knockout mutants.</p> <p>Conclusion</p> <p>The lambda Red-based technique can be used efficiently to generate mutants in <it>P. aeruginosa</it>. The main advantage of this procedure is its rapidity as mutants can be easily obtained in less than a week if the 3-step PCR procedure is used, or in less than three days if the mutation needs to be transferred from one strain to another.</p

Caractérisation de variants phénotypiques de Pseudomonas aeruginosa capable de croître sur de l'hexadécane

Les souches de P. aeruginosa, isolées de sols contaminés aux hydrocarbures, peuvent se développer sur des substrats hydrophobes tels que les alcanes et les hydrocarbures aromatiques polycycliques. Cependant, ces hydrocarbures sont très peu solubles dans l’eau, ce qui entraîne une réduction de la biodisponibilité et de la croissance des bactéries. P. aeruginosa peut réguler sa physiologiques pour s'adapter et développer plus efficacement sur ces hydrocarbures. Ces changements physiologiques incluent des modifications dans l’hydrophobicité de la surface cellulaire, la capacité à adhérer et à former un biofilm, et enfin la production de biosurfactants. Notre laboratoire a montré que la croissance sur des alcanes linéaires liquides tels que l'hexadécane est favorisée chez certaines souches de P. aeruginosa par la formation de variant phénotypiques (dites SCV), qui sont plus adhérents et plus hydrophobes que la forme de phénotype sauvage. La caractérisation des SCV a montré une réduction de la motilité de type swimming et swarming, tandis que la motilité de type twitching a augmenté. Les SCV de la souche 57RP affichent de meilleures capacités dans la formation de biofilms et dans l'adhérence aux hydrocarbures par rapport à la souche sauvage. Nous avons étudié les niveaux intracellulaires du second messager c-di-GMP, qui a été montré être fortement impliqué dans la formation de SCV chez des souches de P. aeruginosa isolées de patients. Nos résultats ont montré une nette augmentation par rapport au type sauvage. Afin de démontrer l’importance du niveau de c-di-GMP chez les SCV, la souche 57RP sauvage a été transformée avec un plasmide portant soit le gène pvrR (impliqué dans la dégradation du c-di-GMP), ou le gène PA14-72420 (impliqué dans la synthèse de c-di-GMP). Les résultats ont indiqué que la souche 57RP(pPA14-72420) cultivée sur de l'hexadécane produit, par rapport à la souche 57RP(ppvrR), un niveau plus élevé de c-di-GMP en plus d'avoir une meilleure croissance sur l'hexadécane et de produire plus de biofilm. Nos résultats démontrent l'importance des changements physiologiques des bactéries pour la croissance sur des composés carbonés très peu solubles

MexEF-OprN Efflux Pump Exports the Pseudomonas Quinolone Signal (PQS) Precursor HHQ (4-hydroxy-2-heptylquinoline)

Bacterial cells have evolved the capacity to communicate between each other via small diffusible chemical signals termed autoinducers. Pseudomonas aeruginosa is an opportunistic pathogen involved, among others, in cystic fibrosis complications. Virulence of P. aeruginosa relies on its ability to produce a number of autoinducers, including 4-hydroxy-2-alkylquinolines (HAQ). In a cell density-dependent manner, accumulated signals induce the expression of multiple targets, especially virulence factors. This phenomenon, called quorum sensing, promotes bacterial capacity to cause disease. Furthermore, P. aeruginosa possesses many multidrug efflux pumps conferring adaptive resistance to antibiotics. Activity of some of these efflux pumps also influences quorum sensing. The present study demonstrates that the MexEF-OprN efflux pump modulates quorum sensing through secretion of a signalling molecule belonging to the HAQ family. Moreover, activation of MexEF-OprN reduces virulence factor expression and swarming motility. Since MexEF-OprN can be activated in infected hosts even in the absence of antibiotic selective pressure, it could promote establishment of chronic infections in the lungs of people suffering from cystic fibrosis, thus diminishing the immune response to virulence factors. Therapeutic drugs that affect multidrug efflux pumps and HAQ-mediated quorum sensing would be valuable tools to shut down bacterial virulence

Unravelling the genome-wide contributions of specific 2-alkyl-4-quinolones and PqsE to quorum sensing in Pseudomonas aeruginosa

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl 4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response

Rhamnolipids: diversity of structures, microbial origins and roles

Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C10-C10), α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoate (Rha-Rha-C10), as well as their mono-rhamnolipid congeners Rha-C10-C10 and Rha-C10. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development

Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

YesN-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach.University of Malaya High Impact Research (HIR) Grant (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027) given to K.G.C. and National Natural Science Foundation of China (no. 81260481) given to H.W

Surface Hardness Impairment of Quorum Sensing and Swarming for Pseudomonas aeruginosa

The importance of rhamnolipid to swarming of the bacterium Pseudomonas aeruginosa is well established. It is frequently, but not exclusively, observed that P. aeruginosa swarms in tendril patterns—formation of these tendrils requires rhamnolipid. We were interested to explain the impact of surface changes on P. aeruginosa swarm tendril development. Here we report that P. aeruginosa quorum sensing and rhamnolipid production is impaired when growing on harder semi-solid surfaces. P. aeruginosa wild-type swarms showed huge variation in tendril formation with small deviations to the “standard” swarm agar concentration of 0.5%. These macroscopic differences correlated with microscopic investigation of cells close to the advancing swarm edge using fluorescent gene reporters. Tendril swarms showed significant rhlA-gfp reporter expression right up to the advancing edge of swarming cells while swarms without tendrils (grown on harder agar) showed no rhlA-gfp reporter expression near the advancing edge. This difference in rhamnolipid gene expression can be explained by the necessity of quorum sensing for rhamnolipid production. We provide evidence that harder surfaces seem to limit induction of quorum sensing genes near the advancing swarm edge and these localized effects were sufficient to explain the lack of tendril formation on hard agar. We were unable to artificially stimulate rhamnolipid tendril formation with added acyl-homoserine lactone signals or increasing the carbon nutrients. This suggests that quorum sensing on surfaces is controlled in a manner that is not solely population dependent