Revisiting the virulence hallmarks of Pseudomonas aeruginosa: a chronicle through the perspective of quorum sensing

Pseudomonas aeruginosa is an opportunistic pathogen and the leading cause of mortality among immunocompromised patients in clinical setups. The hallmarks of virulence in P. aeruginosa encompass six biologically competent attributes that cumulatively drive disease progression in a multistep manner. These multifaceted hallmarks lay the principal foundation for rationalizing the complexities of pseudomonal infections. They include factors for host colonization and bacterial motility, biofilm formation, production of destructive enzymes, toxic secondary metabolites, iron-chelating siderophores and toxins. This arsenal of virulence hallmarks is fostered and stringently regulated by the bacterial signalling system called quorum sensing (QS). The central regulatory functions of QS in controlling the timely expression of these virulence hallmarks for adaptation and survival drive the disease outcome. This review describes the intricate mechanisms of QS in P. aeruginosa and its role in shaping bacterial responses, boosting bacterial fitness. We summarize the virulence hallmarks of P. aeruginosa, relating them with the QS circuitry in clinical infections. We also examine the role of QS in the development of drug resistance and propose a novel antivirulence therapy to combat P. aeruginosa infections. This can prove to be a next-generation therapy that may eventually become refractory to the use of conventional antimicrobial treatments.     

A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced toll pathway in an insect

The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Sp?tzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Sp?tzle processing enzyme (SPE), a terminal enzyme that converts Sp?tzle pro-protein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Sp?tzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Sp?tzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Sp?tzle processing is essential for the PG-dependent Toll signaling pathway.     

Heterologous expression of human paraoxonases in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits biofilm formation and decreases antibiotic resistance

Quorum sensing (QS) regulates virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Human paraoxonases (hPONs) are a family of closely related enzymes with multiple functions, including inactivation of the QS signal molecule in P. aeruginosa. However, there is no direct evidence to show the functions of hPONs on biofilm formation and antibiotic resistance in P. aeruginosa. In the present study, hPONs (hPON1, hPON2, and hPON3) genes were respectively cloned into the pMEKm12 shuttle vector and transformed into P. aeruginosa strain PAO1. Expression of the three recombinant proteins was confirmed by Western blotting, and growth of the recombinant strains was not affected by the hPONs gene expression. Biofilm formation and antibiotics resistance of the hPONs recombinant strains were analyzed. Our results showed that biofilm formation was significantly inhibited in all of the three hPONs recombinant strains. Interestingly, this inhibition can be reverted by addition of the corresponding hPONs polyclonal antibodies in the culture media, further indicating that the inhibition of biofilm formation was due to hPONs protein expression. In addition, we also demonstrated that hPONs expression decreased resistance of P. aeruginosa to gentamicin and ceftazidima, two antibiotics clinically used for the treatment of P. aeruginosa infection.     

Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses

Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and β-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.     

Molecular control of phenoloxidase-induced melanin synthesis in an insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Sp?tzle-processing enzyme is a key enzyme in the production of cleaved Sp?tzle from pro-Sp?tzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Sp?tzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.     

A Spätzle-processing enzyme required for toll signaling activation in Drosophila innate immunity

The Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Sp?tzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Sp?tzle processing during the immune response has remained elusive for a decade. Here, we report a protease, called Sp?tzle-processing enzyme (SPE), required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Sp?tzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes.     

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors’ production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-l-homoserine lactone (C₄–HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

     

铜绿假单胞菌群体感应信号分子PQS的功能多样性研究进展

铜绿假单胞菌(Pseudomonas aeruginosa)是一种革兰氏阴性条件致病菌,可对免疫功能低下或损伤的患者造成持续性感染。铜绿假单胞菌能成功感染离不开其自身产生的毒力因子,而这些毒力因子大多数都受群体感应系统(quorum sensing,QS)调控。铜绿假单胞菌有4个QS系统,分别为las系统、rhl系统、pqs系统和iqs系统。2-庚基-3-羟基-4-喹诺酮(Pseudomonas quinolone signal,PQS)作为铜绿假单胞菌pqs系统的信号分子,不仅能够调控许多毒力因子的表达,也能够影响一些微生物和宿主的多种生理过程。本文总结了PQS多种生物学功能,如介导QS系统、调控生物被膜形成、介导外膜囊泡产生及铁摄取、调节宿主免疫活性、介导细胞毒性作用,以及提供种群保护等。本文旨在突出铜绿假单胞菌PQS的功能多样性,并为PQS新功能研究和抗菌药物的研发提供指导。     

Effect of Cinnamon Oil on Quorum Sensing-Controlled Virulence Factors and Biofilm Formation in Pseudomonas aeruginosa

Quorum sensing (QS) is a system of stimuli and responses in bacterial cells governed by their population density, through which they regulate genes that control virulence factors and biofilm formation. Despite considerable research on QS and the discovery of new antibiotics, QS-controlled biofilm formation by microorganisms in clinical settings has remained a problem because of nascent drug resistance, which requires screening of diverse compounds for anti-QS activities. Cinnamon is a dietary phytochemical that is traditionally used to remedy digestive problems and assorted contagions, which suggests that cinnamon might contain chemicals that can hinder the QS process. To test this hypothesis, the anti-QS activity of cinnamon oil against P. aeruginosa was tested, measured by the inhibition of biofilm formation and other QS-associated phenomena, including virulence factors such as pyocyanin, rhamnolipid, protease, alginate production, and swarming activity. To this end, multiple microscopy analyses, including light, scanning electron and confocal microscopy, revealed the ability of cinnamon oil to inhibit P. aeruginosa PAO1 biofilms and their accompanying extracellular polymeric substances. This work is the first to demonstrate that cinnamon oil can influence various QS-based phenomena in P. aeruginosa PAO1, including biofilm formation.     

Expression and regulation of Spätzle-processing enzyme in Drosophila

The Drosophila melanogaster Toll receptor controls embryonic dorsal-ventral axis formation and is crucial for the innate immune response. In both cases, Toll is activated by the enzymatically cleaved form of its ligand Sp?tzle (Spz). During axis formation, Spz is cleaved by the maternally provided serine protease Easter while the Sp?tzle-processing enzyme (SPE) activates Spz after infection. We confirm the role of SPE in immunity and show that it is a zygotic gene specifically expressed in immune tissues implying that the dual activation of Spz is achieved by differential spatiotemporal expression of two similar but distinct serine proteases.     

Functional Insights from the Crystal Structure of the N-Terminal Domain of the Prototypical Toll Receptor

1. Download : Download high-res image (219KB)
2. Download : Download full-size image

Highlights? The N-terminal cap of Toll adopts a new fold ? It resembles a duplicated beta hairpin structure with three disulphide bonds ? Toll LRRNT cap is not critical for Spätzle binding ? The first 13 LRRs are sufficient for Spätzle binding     

Impact of Azithromycin on the Quorum Sensing-Controlled Proteome of Pseudomonas aeruginosa

The macrolide antibiotic, azithromycin (AZM), has been reported to improve the clinical outcome of cystic fibrosis patients, many of whom are chronically-infected with Pseudomonas aeruginosa. However, the highest clinically-achievable concentrations of this drug are well-below the minimum inhibitory concentration for P. aeruginosa, raising the question of why AZM exhibits therapeutic activity. One possibility that has been raised by earlier studies is that AZM inhibits quorum sensing (QS) by P. aeruginosa. To explicitly test this hypothesis the changes brought about by AZM treatment need to be compared with those associated with specific QS mutants grown alongside in the same growth medium, but this has not been done. In this work, we used quantitative 2D-difference gel electrophoresis and ¹H-NMR spectroscopy footprint analysis to examine whether a range of clinically-relevant AZM concentrations elicited proteomic and metabolomic changes in wild-type cultures that were similar to those seen in cultures of defined QS mutants. Consistent with earlier reports, over half of the AZM-induced spot changes on the 2D gels were found to affect QS-regulated proteins. However, AZM modulated very few protein spots overall (compared with QS) and collectively, these modulated proteins comprised only a small fraction (12–13%) of the global QS regulon. We conclude that AZM perturbs a sub-regulon of the QS system but does not block QS per se. Reinforcing this notion, we further show that AZM is capable of attenuating virulence factor production in another Gram-negative species that secretes copious quantities of exoenzymes (Serratia marcescens), even in the absence of a functional QS system.     

Coordinated regulation of anthranilate metabolism and bacterial virulence by the GntR family regulator MpaR in Pseudomonas aeruginosa

The GntR family regulators are widely distributed in bacteria and play critical roles in metabolic processes and bacterial pathogenicity. In this study, we describe a GntR family protein encoded by PA4132 that we named MpaR (M vfR-mediated P QS and a nthranilate r egulator) for its regulation of Pseudomonas quinolone signal (PQS) production and anthranilate metabolism in Pseudomonas aeruginosa. The deletion of mpaR increased biofilm formation and reduced pyocyanin production. RNA sequencing analysis revealed that the mRNA levels of antABC encoding enzymes for the synthesis of catechol from anthranilate, a precursor of the PQS, were most affected by mpaR deletion. Data showed that MpaR directly activates the expression of mvfR, a master regulator of pqs system, and subsequently promotes PQS production. Accordingly, deletion of mpaR activates the expression of antABC genes, and thus, increases catechol production. We also demonstrated that MpaR represses the rhl quorum-sensing (QS) system, which has been shown to control antABC activity. These results suggested that MpaR function is integrated into the QS regulatory network. Moreover, mutation of mpaR promotes bacterial survival in a mouse model of acute pneumonia infection. Collectively, this study identified a novel regulator of pqs system, which coordinately controls anthranilate metabolism and bacterial virulence in P. aeruginosa.     

Novel thiazolinyl-picolinamide based palladium(II) complex-impregnated urinary catheters quench the virulence and disintegrate the biofilms of uropathogens

Abstract

Pseudomonas aeruginosa and Serratia marcescens are prominent members belonging to the group of ESKAPE pathogens responsible for Urinary Tract Infections (UTI) and nosocomial infections. Both the pathogens regulate several virulence factors, including biofilm formation through quorum sensing (QS), an intercellular communication mechanism. The present study describes the anti-biofilm and QS quenching effect of thiazolinyl-picolinamide based palladium(II) complexes against P. aeruginosa and S. marcescens. Palladium(II) complexes showed quorum sensing inhibitory potential in inhibiting swarming motility behaviour, pyocyanin production and other QS mediated virulence factors in both P. aeruginosa and S. marcescens. In addition, the establishment of biofilms was prevented on palladium (II) coated catheters. Overall, the present study demonstrates that thiazolinyl-picolinamide based palladium (II) complexes will be a promising strategy to combat device-mediated UTI infections.     

Caenorhabditis elegans Senses Bacterial Autoinducers

Pseudomonas aeruginosa uses virulence factors controlled by quorum sensing (QS) to kill Caenorhabditis elegans. Here we show that C. elegans is attracted to the acylated homoserine lactones (AHSLs) that mediate QS in P. aeruginosa. Our data also indicate that C. elegans can distinguish AHSLs and may use them to mediate aversive or attractive learning.     

A Toll-Spätzle pathway in the tobacco hornworm, Manduca sexta

Insects synthesize a battery of antimicrobial peptides (AMPs) and expression of AMP genes is regulated by the Toll and Imd (immune deficiency) pathways in Drosophila melanogaster. Drosophila Toll pathway is activated after Spätzle (Spz) is cleaved by Spätzle processing enzyme (SPE) to release the active C-terminal C106 domain (DmSpz-C106), which then binds to the Toll receptor to initiate the signaling pathway and regulate expression of AMP genes such as drosomycin. Toll and Spz genes have been identified in other insects, but interaction between Toll and Spz and direct evidence for a Toll-Spz pathway in other insect species have not been demonstrated. Our aim is to investigate a Toll-Spz pathway in Manduca sexta, and compare M. sexta and D. melanogaster Toll-Spz pathways. Co-immunoprecipitation (Co-IP) assays showed that MsToll^ecto (the ecto-domain of M. sexta Toll) could interact with MsSpz-C108 (the active C-terminal C108 domain of M. sexta Spz) but not with full-length MsSpz, and DmToll^ecto could interact with DmSpz-C106 but not DmSpz, suggesting that Toll receptor only binds to the active C-terminal domain of Spz. Co-expression of MsToll-MsSpz-C108, but not MsToll-MsSpz, could up-regulate expression of drosomycin gene in Drosophila S2 cells, indicating that MsToll-MsSpz-C108 complex can activate the Toll signaling pathway. In vivo assays showed that activation of AMP genes, including cecropin, attacin, moricin and lebocin, in M. sexta larvae by purified recombinant MsSpz-C108 could be blocked by pre-injection of antibody to MsToll, further confirming a Toll-Spz pathway in M. sexta, a lepidopteran insect.