Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43

A bacterial strain, which based on the sequences of its 16S rRNA, gyrB, catA, and qsdA genes, was identified as a Rhodococcus sp. closely related to Rhodococcus erythropolis, was isolated from soil by enrichment on the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogen Pseudomonas aeruginosa. The isolate, termed Rhodococcus sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of P. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably, Rhodococcus sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite of P. aeruginosa to a quorum sensing signal molecule.     

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.     

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。     

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.     

Growth phase-differential quorum sensing regulation of anthranilate metabolism in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas quinolone signal (PQS) plays a role in the regulation of virulence genes and it is intertwined in the las/rhl quorum sensing (QS) circuits of Pseudomonas aeruginosa. PQS is synthesized from anthranilate by pqsA-D and pqsH whose expression is influenced by the las/rhl systems. Since anthranilate can be degraded by functions of antABC and catBCA, PQS synthesis might be regulated by the balance between the expression of the pqsA-D/phnAB, pqsH, antABC, and catBCA gene loci. antA and catA are repressed by LasR during log phase and activated by RhlR in late stationary phase, whereas pqsA-E/phnAB is activated by LasR in log phase and repressed by RhlR. QscR represses both but each repression occurs in a different growth phase. This growth phase-differential regulation appears to be accomplished by the antagonistic interplay of LasR, RhlR, and QscR, mediated by two intermediate regulators, AntR and PqsR, and their cofactors, anthranilate and PQS, where the expressions of antR and pqsR and the production of anthranilate and PQS are growth phase-differentially regulated by QS systems. Especially, the anthranilate level increases in an RhlR-dependent manner at late stationary phase. From these results, we suggest that RhlR and LasR regulate the anthranilate metabolism in a mutually antagonistic and growth phase-differential manner by affecting both the expressions and activities of AntR and PqsR, and that QscR also phase-differentially represses both LasR and RhlR functions in this regulation.     

Quorum sensing by 2-alkyl-4-quinolones in Pseudomonas aeruginosa and other bacterial species

Pseudomonas aeruginosa produces the cell-to-cell signal molecule 2-heptyl-3-hydroxy-4-quinolone (The Pseudomonas quinolone signal; PQS), which is integrated within a complicated quorum sensing signaling system. PQS belongs to the family of 2-alkyl-4-quinolones (AQs), which have been previously described for their antimicrobial activities. PQS is synthesized via the pqsABCDE operon which is responsible for generating multiple AQs including 2-heptyl-4-quinolone (HHQ), the immediate PQS precursor. In addition, PQS signaling plays an important role in P. aeruginosa pathogenesis because it regulates the production of diverse virulence factors including elastase, pyocyanin and LecA lectin in addition to affecting biofilm formation. Here, we summarize the most recent findings on the biosynthesis and regulation of PQS and other AQs including the discovery of AQs in other bacterial species.     

The Stringent Response Modulates 4-Hydroxy-2-Alkylquinoline Biosynthesis and Quorum-Sensing Hierarchy in Pseudomonas aeruginosa

As a ubiquitous environmental organism and an important human pathogen, Pseudomonas aeruginosa readily adapts and responds to a wide range of conditions and habitats. The intricate regulatory networks that link quorum sensing and other global regulators allow P. aeruginosa to coordinate its gene expression and cell signaling in response to different growth conditions and stressors. Upon nutrient transitions and starvation, as well as other environmental stresses, the stringent response is activated, mediated by the signal (p)ppGpp. P. aeruginosa produces a family of molecules called HAQ (4-hydroxy-2-alkylquinolines), some of which exhibit antibacterial and quorum-sensing signaling functions and regulate virulence genes. In this study, we report that (p)ppGpp negatively regulates HAQ biosynthesis: in a (p)ppGpp-null (ΔSR) mutant, HHQ (4-hydroxyl-2-heptylquinoline) and PQS (3,4-dihydroxy-2-heptylquinoline) levels are increased due to upregulated pqsA and pqsR expression and reduced repression by the rhl system. We also found that (p)ppGpp is required for full expression of both rhl and las AHL (acyl-homoserine lactone) quorum-sensing systems, since the ΔSR mutant has reduced rhlI, rhlR, lasI, and lasR expression, butanoyl-homoserine lactone (C₄-HSL) and 3-oxo-dodecanoyl-homoserine lactone (3-oxo-C₁₂-HSL) levels, and rhamnolipid and elastase production. Furthermore, (p)ppGpp significantly modulates the AHL and PQS quorum-sensing hierarchy, as the las system no longer has a dominant effect on HAQ biosynthesis when the stringent response is inactivated.     

Pseudomonas quinolone signal induces organelle stress and dysregulates inflammation in human macrophages

Pseudomonas quinolone signal (PQS) is a quorum-sensing molecule associated with Pseudomonas aeruginosa that regulates quorum sensing, extracellular vesicle biogenesis, iron acquisition, and the secretion of virulence factors. PQS has been shown to have immunomodulatory effects on the host. It induces oxidative stress, modulates cytokine levels, and activates regulated cell death in the host. In this study, we investigated the effects of PQS (10 μM) on host organelle dynamics and dysfunction in human macrophages at the interphase of endoplasmic reticulum (ER), mitochondria, and lysosome. This study showed that PQS increases cytosolic Ca⁺² levels and elevates ER stress, as evidenced by increased expression of BiP and activation of the PERK-CHOP axis of unfolded protein response (UPR). Moreover, PQS also negatively affects mitochondria by disrupting mitochondrial membrane potential and increasing mitochondrial ROS generation (mROS). Additionally, PQS stimulation decreased the number of acridine orange-positive lysosomes, indicating lysosomal destabilization. Furthermore, PQS-induced lysosomal destabilization also induces overexpression of the lysosomal stress-responsive gene TFEB. Besides organelle dysfunction, PQS dysregulates inflammation-related genes by upregulating NLRC4, TMS1, and Caspase 1 while downregulating NLRP3 and IL-1β. Also, PQS increases gene expression of pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ). In conclusion, our findings suggest that PQS negatively affects human macrophages by interfering with organelle function and dysregulating inflammatory response. Consequently, this study provides crucial insight into PQS-driven macrophage dysfunction and may contribute to a better understanding of Pseudomonas aeruginosa-associated infections.     

Synthesis and biotransformation of 2-alkyl-4(1<Emphasis Type="Italic">H</Emphasis>)-quinolones by recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the “Pseudomonas quinolone signal” (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 μM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C₃- to C₁₃-saturated and C_7:1 to C_13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and β carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C₅-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in >90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.     

Stress-Induced Outer Membrane Vesicle Production by Pseudomonas aeruginosa

As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.     

The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing

In iron-replete environments, the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein represses expression of two small regulatory RNAs encoded by prrF1 and prrF2. Here we describe the effects of iron and PrrF regulation on P. aeruginosa physiology. We show that PrrF represses genes encoding enzymes for the degradation of anthranilate (i.e. antABC), a precursor of the Pseudomonas quinolone signal (PQS). Under iron-limiting conditions, PQS production was greatly decreased in a DeltaprrF1,2 mutant as compared with wild type. The addition of anthranilate to the growth medium restored PQS production to the DeltaprrF1,2 mutant, indicating that its defect in PQS production is a consequence of anthranilate degradation. PA2511 was shown to encode an anthranilate-dependent activator of the ant genes and was subsequently renamed antR. AntR was not required for regulation of antA by PrrF but was required for optimal iron activation of antA. Furthermore, iron was capable of activating both antA and antR in a DeltaprrF1,2 mutant, indicating the presence of two distinct yet overlapping pathways for iron activation of antA (AntR-dependent and PrrF-dependent). Additionally, several quorum-sensing regulators, including PqsR, influenced antA expression, demonstrating that regulation of anthranilate metabolism is intimately woven into the quorum-sensing network of P. aeruginosa. Overall, our data illustrate the extensive control that both iron regulation and quorum sensing exercise in basic cellular physiology, underlining how intermediary metabolism can affect the regulation of virulence factors in P. aeruginosa.