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Relative molar proportions ( pie charts ) and quantitative profiling ( bar graphs ) of AHLs and AQs produced by P. aeruginosa wild-type strain grown in LB and CDM growth medium
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An LC-MS/MS method, using positive mode electrospray ionization, for the simultaneous, quantitative and targeted profiling of the N-acyl-L-homoserine lactone (AHL) and 2-alkyl 4-(1H)-quinolone (AQ) families of bacterial quorum-sensing signaling molecules (QSSMs) is presented. This LC-MS/MS technique was applied to determine the relative molar ratio...
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... aeruginosa produced a wide range of QSSMs, the nature of which varies in acyl/alkyl chain length as well as the level of substitution. When grown in LB, the P. aeruginosa PAO1 parent strain produced primarily C4-HSL (31 μ M) and 3-oxo-C12-HSL (0.5 μ M) but also C6-HSL (0.9 μ M) and 3-OH-C4-HSL (0.5 μ M; Fig. 4, Table 3). These profiles and concentrations of AHLs are in line with those found in a previously published study [35]. A similar profile was observed following growth in CDM although the overall AHL concentrations were lower than in LB: C4- HSL (6 μ M) and 3-oxo-C12-HSL (1.4 μ M) C6-HSL (0.7 μ M) and 3-OH-C4-HSL (0.1 μ M; Fig. 4, Table 3). Similarly, total AQ concentrations in CDM (5.6 μ M) were lower than those observed in LB (9.4 μ M) with a notable 10-fold higher concentration of PQS in LB compared with CDM. The concentration of PQS generally documented after growth in LB corresponds to concentrations 10 to 100- times higher (5 – 10 μ M) [19] than in CDM. In LB batch culture, PQS has been detected in the logarithmic phase and a P. aeruginosa culture contains 5 – 10 μ M PQS which increased up to 25 μ M in stationary phase [19]. A recent study showed that the AQ biosynthetic pathway and the anthranilate degradation pathway, which both feed into the TCA cycle for energy metabolism, draw from the same pool of intracellular metabolite [44, 45]. Therefore, in CDM, the metabolite pools directing PQS synthesis may be drawn into the TCA cycle to sustain cell growth at the cost of AQ synthesis. Analysis of the QSSM profiles of mutants of P. aeruginosa grown in CDM showed that disruption of one QS system (AHL or AQ biosynthetic genes) impacts on the synthesis of the QSSMs from other QS systems. Examples of extracted ion LC-MS/MS chromatograms are given in Electronic Supplementary Material Fig. S1. The full data from this series of experiments is shown in Electronic Supplementary Material Table S1 and Fig. 5. The disruption of the pqsA gene resulted in a twofold increase in C4- HSL and C6-HSL and a significant reduction in 3-oxo- C12-HSL when compared with the wild type. Furthermore, a mutation in rhlI resulted in a large increase in HQNO, NQNO, and PQS levels while a mutation in lasI resulted in a significantly increased level of HHQ, NHQ, the disap- pearance of PQS congeners and a large reduction in C4- HSL levels (Fig. 5, Electronic Supplementary Material Table S1). It has been previously established that both the las and rhl systems exert an influence on the pqs system and that the pqs system affects the synthesis of C4-HSL and 3-oxo-C12-HSL (reviewed by Heeb et al. [46]. McKnight et al. [20] showed that PQS positively regulates rhlI expression, thus influencing C4-HSL accumulation. It has also been observed that pqsABCDE expression depends on the ratio between the two AHL molecules, 3-oxo-C12- HSL having a positive effect, and C4-HSL having a negative effect [47] this has also been corroborated by [48] who showed that whereas LasR has a positive impact on pqsR expression and hence AQ production, RhlI has the opposite effect. The increase in HHQ and NHQ in the lasI mutant can be explained by the positive control of pqsH (which encodes the mono-oxygenase required for the conversion of HHQ and NHQ to PQS and C9-PQS) by LasR/3-oxo- C12-HSL. Similar results have been obtained previously [49 – 51]). A selective and rapid method for the simultaneous analysis of the two main classes of QSSMs has been developed, validated and shown to be applicable to obtain good quality experimental data from P. aeruginosa . In addition to this application, the method has been successfully applied to measure AHL and AQ profiles in both complex and defined culture media under a range of experimental situations. Although this methodology was conceived for profiling studies in relation to the biosynthesis of QSSMs, the methodology could be easily modified to facilitate studies on biological samples other than bacterial cultures. We have started to exploit this technology for the analysis QSSM levels in the body fluids of patients with cystic fibrosis who are colonized with P. aeruginosa or Burkholderia spp., both of which produce complex mixtures of AHLs and AQs [23, 52]. Therefore, the technology presented in this manuscript will be a valuable investigative tool for metabolic profiling in many environments including the ...
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... aeruginosa produced a wide range of QSSMs, the nature of which varies in acyl/alkyl chain length as well as the level of substitution. When grown in LB, the P. aeruginosa PAO1 parent strain produced primarily C4-HSL (31 μ M) and 3-oxo-C12-HSL (0.5 μ M) but also C6-HSL (0.9 μ M) and 3-OH-C4-HSL (0.5 μ M; Fig. 4, Table 3). These profiles and concentrations of AHLs are in line with those found in a previously published study [35]. A similar profile was observed following growth in CDM although the overall AHL concentrations were lower than in LB: C4- HSL (6 μ M) and 3-oxo-C12-HSL (1.4 μ M) C6-HSL (0.7 μ M) and 3-OH-C4-HSL (0.1 μ M; Fig. 4, Table 3). Similarly, total AQ concentrations in CDM (5.6 μ M) were lower than those observed in LB (9.4 μ M) with a notable 10-fold higher concentration of PQS in LB compared with CDM. The concentration of PQS generally documented after growth in LB corresponds to concentrations 10 to 100- times higher (5 – 10 μ M) [19] than in CDM. In LB batch culture, PQS has been detected in the logarithmic phase and a P. aeruginosa culture contains 5 – 10 μ M PQS which increased up to 25 μ M in stationary phase [19]. A recent study showed that the AQ biosynthetic pathway and the anthranilate degradation pathway, which both feed into the TCA cycle for energy metabolism, draw from the same pool of intracellular metabolite [44, 45]. Therefore, in CDM, the metabolite pools directing PQS synthesis may be drawn into the TCA cycle to sustain cell growth at the cost of AQ synthesis. Analysis of the QSSM profiles of mutants of P. aeruginosa grown in CDM showed that disruption of one QS system (AHL or AQ biosynthetic genes) impacts on the synthesis of the QSSMs from other QS systems. Examples of extracted ion LC-MS/MS chromatograms are given in Electronic Supplementary Material Fig. S1. The full data from this series of experiments is shown in Electronic Supplementary Material Table S1 and Fig. 5. The disruption of the pqsA gene resulted in a twofold increase in C4- HSL and C6-HSL and a significant reduction in 3-oxo- C12-HSL when compared with the wild type. Furthermore, a mutation in rhlI resulted in a large increase in HQNO, NQNO, and PQS levels while a mutation in lasI resulted in a significantly increased level of HHQ, NHQ, the disap- pearance of PQS congeners and a large reduction in C4- HSL levels (Fig. 5, Electronic Supplementary Material Table S1). It has been previously established that both the las and rhl systems exert an influence on the pqs system and that the pqs system affects the synthesis of C4-HSL and 3-oxo-C12-HSL (reviewed by Heeb et al. [46]. McKnight et al. [20] showed that PQS positively regulates rhlI expression, thus influencing C4-HSL accumulation. It has also been observed that pqsABCDE expression depends on the ratio between the two AHL molecules, 3-oxo-C12- HSL having a positive effect, and C4-HSL having a negative effect [47] this has also been corroborated by [48] who showed that whereas LasR has a positive impact on pqsR expression and hence AQ production, RhlI has the opposite effect. The increase in HHQ and NHQ in the lasI mutant can be explained by the positive control of pqsH (which encodes the mono-oxygenase required for the conversion of HHQ and NHQ to PQS and C9-PQS) by LasR/3-oxo- C12-HSL. Similar results have been obtained previously [49 – 51]). A selective and rapid method for the simultaneous analysis of the two main classes of QSSMs has been developed, validated and shown to be applicable to obtain good quality experimental data from P. aeruginosa . In addition to this application, the method has been successfully applied to measure AHL and AQ profiles in both complex and defined culture media under a range of experimental situations. Although this methodology was conceived for profiling studies in relation to the biosynthesis of QSSMs, the methodology could be easily modified to facilitate studies on biological samples other than bacterial cultures. We have started to exploit this technology for the analysis QSSM levels in the body fluids of patients with cystic fibrosis who are colonized with P. aeruginosa or Burkholderia spp., both of which produce complex mixtures of AHLs and AQs [23, 52]. Therefore, the technology presented in this manuscript will be a valuable investigative tool for metabolic profiling in many environments including the ...
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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...
Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates.
Using bacterial...
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... Ethylenediaminetetraacetic acid (EDTA), a metal chelator with high formation constants, was added to the aqueous mobile phase solution to continuously bind the trace metal cations in the HPLC system. [34][35] Figure S2(a) in Supporting Information A shows the chromatogram of murexide with a well-defined peak shape when using the mobile phase with 2 mM EDTA. Thus, 2 mM EDTA was added to the aqueous solution before mixing with the methanol (see "Stopped-flow HPLC method" and "Kinetic measurements in the reversedphase C8 column") and used for both on-column and batch kinetics. ...
A kinetic process can sometimes be observed during a chromatographic separation. The chemical reaction is coupled with the rapid distribution of the reacting compounds between the mobile and stationary phases. The use of the pre‐equilibrium approximation enables the derivation of the rate law for the chemical reaction(s) taking place in the mobile and/or stationary phase. A simple procedure is proposed for writing down this rate equation. It can be applied to any on‐column reaction, whether reversible or irreversible. For first‐ or pseudo‐first‐order kinetics, integration of the rate equation gives an exponential function of concentration with respect to time. To demonstrate the application of the proposed method, the kinetics of the decomposition of murexide in a reversed‐phase C8 column was studied. Murexide decomposes through a fast reversible protonation process to give purpuric acid, which then decomposes irreversibly. The stopped‐flow HPLC technique was used to obtain the entire kinetic profile for the on‐column reaction. Additionally, the kinetics was measured off‐column using the same mobile phase. The values of the kinetic parameters obtained from the batch experiments enabled the determination of all kinetic parameters of the on‐column reaction using non‐linear least squares fitting of the derived rate equation.
... These bacteria are also common adhesion and biofilm formation models [17]. The w.t. and DflgF were purchased from the Washington University Manoil laboratory mutant library [18]; the DpqsA and DlasI mutants [19] were kindly provided by Professor Miguel Camara of Nottingham University; the DfliC and DpilA mutants [20] were kindly provided by Professor Martin Welch of Cambridge University. All P. aeruginosa strains tested were transformed by electroporation with the pMRP9-1 plasmid, for constitutive expression of GFPmut2 and Carbenicillin resistance [21], kindly provided by Professor Ehud Banin of the Bar Ilan University. ...
Bacterial adhesion to tissue is the starting point for many pathogenic processes and beneficial interactions. The dynamics and speed of adhesion (minutes) make high-resolution temporal kinetic data important, but this capability is absent from the current toolset. We present a high-throughput method with a second-to-minute kinetic resolution, testing the adhesion of Pseudomonas aeruginosa PAO1 wild-type, flagella-, pili-, and quorum-sensing mutants to human embryonic kidney (HEK293) cells. Adhesion rates were in good correlation with HEK293 confluence, and the ways in which various bacterial mutations modified adhesion patterns are in agreement with the published literature. This simple assay can facilitate drug screening and treatment development as well as provide a better understanding of the interactions of pathogenic and probiotic bacteria with tissues, allowing the design of interventions and prevention treatments.
... The mobile phases, conditions, and gradient used for LC-MS/MS analysis are shown in Supplementary Table S2, and Supplementary Table S3 summarizes the MS parameter settings. Metabolomic identification of autoinducers in bacterial extracts was performed by untargeted Q3 + scan and targeted neutral loss scan based on the characteristic fragmentation patterns of the lactone ring at m/z 102.1 for AHLs [12] . Metabolite expression was quantified as the area under the curve formed during multiple reaction monitoring (MRM) of identified compounds. ...
The goal of this study was to determine the effects of phenolic extracts from grape (GrPE), pomegranate (PoPE), and persimmon (PePE) by-products on bacterial virulence activities such as biofilms, motility, energy-dependent efflux pumps, and β-lactamase activity, which are primarily modulated by quorum sensing (QS), defining their potential applications. The microdilution method was used to determine the minimum inhibitory concentration (MIC) and sub-inhibitory concentrations (SICs) of the extracts against reference pathogenic bacteria. The antibacterial mode of action was determined by labelling bacterial cells in in vivo cell-tracking experiments. Antibiograms showed that PoPE inhibited bacteria at lower concentrations, while PePE had a stronger effect against Klebsiella pneumoniae. Both extracts caused significant cell membrane damage (CMD), while GrPE did not. At SICs, all extracts showed anti-QS activity, especially PePE, which inhibited violacein and pyocyanin production at 1/128 × MIC. Additionally, QS autoinducers found in Chromobacterium violaceum and Pseudomonas aeruginosa were modulated by the extracts, PePE showed the highest modulation. Antibiofilm assays revealed that GrPE, at MIC and 2 × MIC, acted as a potent antibiofilm agent against biofilms of Pseudomonas putida, Bacillus cereus, and Staphylococcus aureus, which was related to disruption of swarming motility by GrPE. All extracts, especially PoPE, exerted a potent effect against the activation of efflux pumps of P. aeruginosa, as well as β-lactamase activity in K. pneumoniae. Results suggest that the anti-virulence potential of the extracts may be related to their effect as extracellular autoinducer modulators. This study allowed to define potential applications of these extracts.
... When required the medium was supplemented with 1 mM isopropyl-b-D-1-thiogalactopyranoside (IPTG). All AQs were synthesized in-house as described by Ortori and collaborators (Ortori et al., 2011). Stock solutions of AQs (10 mM) were prepared in MeOH. ...
... AQs produced by P. aeruginosa PAO1, the corresponding isogenic mutants, and CF lytic strains carrying the pME6032 or pME-pqsL plasmids were quantified by LC-MS/MS analysis, as previously described (Ortori et al., 2011). Briefly, 1 mL of filtered supernatant from overnight cultures grown at 37°C in LB or in LB supplemented with 1 mM IPTG was extracted with ethyl acetate, dried under vacuum, and redissolved in methanol prior to LC-MS/ MS. ...
... The PAO1 DpqsAHL strain showed visible lysis in the presence of HHQ and NHQ, but not when treated with PHQ and UHQ, or with methanol as control ( Figure 2D). Since HHQ and NHQ are produced by wild type P. aeruginosa at similar concentrations Ortori et al., 2011), it is likely that the lytic strains DpqsL and DpqsHL accumulate both NHQ and HHQ, and hence that these Series A congeners both contribute to autolysis in the strains impaired in HQNO synthesis. However, for simplicity and considering that most QS studies focus on HHQ as the primary P. aeruginosa Series A congener and precursor of PQS, only HHQ was considered in further analyses. ...
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.
... Concentrations ranging from 5 to 31 µM for C4-HSL and 0.5-15 µM for 3OC12-HSL in cell-free culture supernatants have been reported [10], while EC 50 s ranging from 1 to 8 μM for RhlR/C4-HSL and 10-139 nM for LasR/3OC12-HSL have been determined for their activation [11][12][13]. RhlI and LasI are also responsible for the biosynthesis of a range of minor AHLs with different acyl chain lengths and 3-position substituents [14]. The function of these is not clear, although some are weaker activators of LasR and RhlR than 3OC12-HSL and C4-HSL, while others have been reported to activate the orphan P. aeruginosa LuxR homologue QscR, which shows a more relaxed specificity for AHLs [15]. ...
... LuxR homologue-independent responses to AHLs have also been described [16]. P. aeruginosa also produces diverse AQs [14,17], which possess antimicrobial, cytotoxic, iron chelating, immune modulatory and QS signalling activities [4,5,18]. These are characterized by the presence or absence of a 3-hydroxyl group, variations in the length of the 2-alkylside chain or N-oxidation [14,17]. ...
... P. aeruginosa also produces diverse AQs [14,17], which possess antimicrobial, cytotoxic, iron chelating, immune modulatory and QS signalling activities [4,5,18]. These are characterized by the presence or absence of a 3-hydroxyl group, variations in the length of the 2-alkylside chain or N-oxidation [14,17]. With respect to the AQs, the major QS signals are the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1 H)-quinolone; Fig. 1] and its immediate biosynthetic precursor, 2-heptyl-4-hydroxyquinoline (HHQ; Fig. 1). ...
In Pseudomonas aeruginosa, quorum sensing (QS) depends on an interconnected regulatory hierarchy involving the Las, Rhl and Pqs systems, which are collectively responsible for the co-ordinated synthesis of a diverse repertoire of N-acylhomoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs). Apparent population density-dependent phenomena such as QS may, however, be due to growth rate and/or nutrient exhaustion in batch culture. Using continuous culture, we show that growth rate and population density independently modulate the accumulation of AHLs and AQs such that the highest concentrations are observed at a slow growth rate and high population density. Carbon source (notably succinate), nutrient limitation (C, N, Fe, Mg) or growth at 25 °C generally reduces AHL and AQ levels, except for P and S limitation, which result in substantially higher concentrations of AQs, particularly AQ N-oxides, despite the lower population densities achieved. Principal component analysis indicates that ~26 % variation is due to nutrient limitation and a further 30 % is due to growth rate. The formation of N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) turnover products such as the ring opened form and tetramic acid varies with the limiting nutrient limitation and anaerobiosis. Differential ratios of N-butanoyl-homoserine lactone (C4-HSL), 3OC12-HSL and the AQs as a function of growth environment are clearly apparent. Inactivation of QS by mutation of three key genes required for QS signal synthesis (lasI, rhlI and pqsA) substantially increases the concentrations of key substrates from the activated methyl cycle and aromatic amino acid biosynthesis, as well as ATP levels, highlighting the energetic drain that AHL and AQ synthesis and hence QS impose on P. aeruginosa.
... This study shows that the D-enantiomer also is produced. [33,34]. Of these, O-C6 and O-C8 were not detected in this study. ...
... Interestingly, of the two D-diastereomers in H-C6, only D2-H-C6 was detected. L-A-C4 was the highest produced N-HL for P. aeruginosa, which also is consistent with previous studies [33,34]. Remarkably, D-A-C4 and L-A-C6 were produced in roughly equivalent amounts (both~10 ng/mL) and were the second most prevalent N-HLs. ...
Quorum Sensing allows bacteria to sense their population density via diffusible N-acyl homoserine lactone (N-HL) signaling molecules. Upon reaching a high enough cell density, bacteria will collectively exhibit a phenotype. Until recently, methods used for detection of N-HLs have not considered the chirality of these molecules and it was assumed that only the L-enantiomer was produced by bacteria. The production and effects of D-N-HLs have rarely been studied. In this work, the temporal production of D-N-HLs by the plant pathogen Pecto-bacterium atrosepticum and the human pathogen Pseudomonas aeruginosa are reported. Both bacteria produced D-N-HLs in significant amounts and in some cases their concentrations were higher than other low abundance L-N-HLs. Previously unreported D-enantiomers of N-3-oxoacyl and N-3-hydroxyacyl homoserine lactones were detected in P. atrosepticum. Interestingly, L-N-HLs produced in the lowest concentrations had relatively higher amounts of their corresponding D-enantiomers. Potential sources of D-N-HLs and their significance are considered.
... One milliliter of culture fluid was centrifuged at 12,000 rpm for 2 min; the supernatant was filtered through a 0.2 μm Millex Syring Filter and then mixed with an equal volume of methanol. The crude extract was subjected to LC-MS/MS as previously reported 52,53 . All MS experiments in this study were performed on an AB SCIEX QTRAP 4500 (Applied Biosystem). ...
Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections. The strong capacity of P. aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure. The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts. Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs. In this study, a high-throughput transposon-insertion site sequencing (Tn-seq) approach was employed to identify novel P. aeruginosa biofilm genetic determinants. When analyzing the novel biofilm regulatory genes, we found that the cell division factor ZapE (PA4438) controls the P. aeruginosa pqs quorum sensing system. The ∆zapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) had been reduced. Further biochemical analysis showed that ZapE interacts with PqsH, which encodes the synthase that converts 2-heptyl-4-quinolone (HHQ) to PQS. In addition, site-directed mutagenesis of the ATPase active site of ZapE (K72A) abolished the positive regulation of ZapE on PQS signaling. As ZapE is highly conserved among the Pseudomonas group, our study suggests that it is a potential drug target for the control of Pseudomonas infections.
... One milliliter of culture fluid was centrifuged at 12,000 rpm for 2 min; the supernatant was filtered through a 0.2 μm Millex Syring Filter and then mixed with an equal volume of methanol. The crude extract was subjected to LC-MS/MS as previously reported 52,53 . All MS experiments in this study were performed on an AB SCIEX QTRAP 4500 (Applied Biosystem). ...
Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections. The strong capacity of P. aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure. The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts. Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs. In this study, a high-throughput transposon-insertion site sequencing (Tn-seq) approach was employed to identify novel P. aeruginosa biofilm genetic determinants. When analyzing the novel biofilm regulatory genes, we found that the cell division factor ZapE (PA4438) controls the P. aeruginosa pqs quorum sensing system. The ΔzapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) had been reduced. Further biochemical analysis showed that ZapE interacts with PqsH, which encodes the synthase that converts 2-heptyl-4-quinolone (HHQ) to PQS. In addition, site-directed mutagenesis of the ATPase active site of ZapE (K72A) abolished the positive regulation of ZapE on PQS signaling. As ZapE is highly conserved among the Pseudomonas group, our study suggests that it is a potential drug target for the control of Pseudomonas infections. Impact statement The drug-resistant biofilm-associated infections caused by Pseudomonas aeruginosa pose critical challenges for clinical treatment, calling for novel antimicrobial strategies targeting biofilm formation mechanisms. Previous investigations of biofilm formation revealed the critical regulatory roles of quorum sensing. In this study, we applied a high-throughput method to identify novel genetical determinants of P. aeruginosa biofilm formation. We found ZapE (PA4438), a cell division factor and an ATPase, that played an important role in regulating P. aeruginosa biofilm formation through the pqs quorum sensing system. Our findings provide a collection of data supporting the concept that ZapE could be a feasible target for antibiofilm and antivirulence approaches.
... Since LasR-3-oxo-C 12 -HSL stimulates both PQS and Rhl QS machinery, these systems represent a hierarchical activation cascade that is triggered by attainment of a threshold cell density. On the other hand, a few studies have reported the presence of other AHLs in P. aeruginosa cultures including C 16 -HSL, C 6 -HSL, C 8 -HSL, 3-oxo-C 10 -HSL and C 18 -HSL, at low concentrations in the AHL profile [23]. Furthermore, studies have reported the detection of C 6 -HSL, C 8 -HSL, C 10 -HSL, 3-oxo-C 6 -HSL and 3-oxo-C 10 -HSL from sputum samples colonised by P. aeruginosa of cystic fibrosis patients [24,25]. ...
... The molecular dynamic simulations depicted that 3-oxo-C 10 -HSL formed a stable complex with LasR compared to 3-oxo-C 12 -HSL by establishing strong hydrogen and hydrophobic interactions. Ortori et al. (2011) reported that 3-oxo-C 10 -HSL was identified in P. aeruginosa culture in LC MS/MS analysis [23]. The docking analysis and molecular simulations of 3-oxo-C 10 -HSL for LasR and 3-oxo-C 6 -HSL for RhlR demonstrated that it has a pleotropic role in QS mechanism. ...
... The molecular dynamic simulations depicted that 3-oxo-C 10 -HSL formed a stable complex with LasR compared to 3-oxo-C 12 -HSL by establishing strong hydrogen and hydrophobic interactions. Ortori et al. (2011) reported that 3-oxo-C 10 -HSL was identified in P. aeruginosa culture in LC MS/MS analysis [23]. The docking analysis and molecular simulations of 3-oxo-C 10 -HSL for LasR and 3-oxo-C 6 -HSL for RhlR demonstrated that it has a pleotropic role in QS mechanism. ...
Pseudomonas aeruginosa is an opportunistic pathogen, having complicated quorum sensing (QS) system utilizing multiple signals and receptors to coordinate virulence and pathogenicity. N-acyl-homoserine-lactones (AHLs) are the most common autoinducers responsible for regulation of QS-mediated virulence gene expression. There are four QS systems in P. aeruginosa among which the LasI/R and RhlI/R systems are regulated by 3-oxo-C12-HSL and C4-HSL respectively. They play a major role in host-associated pathogenesis. The LasR and RhlR binding specificity to cognate or non-cognate HSLs influences the QS-mediated responses. Here, we used computational approaches to consolidate the interaction of different types of HSLs produced by P. aeruginosa with LasR and RhlR receptors. To explore the binding affinity, fourteen different AHLs were subjected for molecular docking analysis with LasR and RhlR receptors. The RhlR was modelled using MMseqs2 in ColabFold: Alpha fold 2. Further, to validate the stability and interaction mechanism, molecular dynamic simulations was performed with the top docked six HSLs for 100 ns. In docking results, apart from 3-oxo-C12-HSL and C4-HSL, other HSLs such as C16-HSL and C6-HSL showed better binding affinity towards LasR and RhlR proteins, respectively. Further validation by molecular dynamic simulations showed that 3-oxo-C10-HSL and 3-oxo-C6-HSL formed stable complex with LasR and RhlR, respectively. Our comprehensive in silico study results may provide promising targets for development of anti-QS drugs against Las/Rhl QS systems.
... To corroborate this, we analyzed the soluble fraction of lysate from E. coli overexpressing RhlR at increasing concentrations of exogenously added C4-HSL in the absence or presence of PqsE overexpression. Western blot analysis demonstrates that only minute amounts of soluble RhlR are detected without C4-HSL and that PqsE enhances RhlR solubility up to at least 50 µM C4-HSL, a concentration corresponding to the higher C4-HSL levels reported for P. aeruginosa [34][35][36] . The amount of RhlR in whole cell extracts, on the other hand, remained unchanged, showing that C4-HSL and PqsE indeed enhance the solubility but not the total amount of RhlR (Fig. 2a, Supplementary Fig. 1a). ...
Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa , with the rhl circuit playing the leading role in late and chronic infections. The majority of traits controlled by rhl transcription factor RhlR depend on PqsE, a dispensable thioesterase in Pseudomonas Quinolone Signal (PQS) biosynthesis that interferes with RhlR through an enigmatic mechanism likely involving direct interaction of both proteins. Here we show that PqsE and RhlR form a 2:2 protein complex that, together with RhlR agonist N -butanoyl-L-homoserine lactone (C4-HSL), solubilizes RhlR and thereby renders the otherwise insoluble transcription factor active. We determine crystal structures of the complex and identify residues essential for the interaction. To corroborate the chaperone-like activity of PqsE, we design stability-optimized variants of RhlR that bypass the need for C4-HSL and PqsE in activating PqsE/RhlR-controlled processes of P. aeruginosa . Together, our data provide insight into the unique regulatory role of PqsE and lay groundwork for developing new P. aeruginosa -specific pharmaceuticals.
