Pseudomonas aeruginosa Isolates from Water Samples of the Gulf of Mexico Show Similar Virulence Properties but Different Antibiotic Susceptibility Profiles than Clinical Isolates.

Pseudomonas aeruginosa is an opportunistic pathogen found in a wide variety of environments, including soil, water, and habitats associated with animals, humans, and plants. From a One Health perspective, which recognizes the interconnectedness of human, animal, and environmental health, it is important to study the virulence characteristics and antibiotic susceptibility of environmental bacteria. In this study, we compared the virulence properties and the antibiotic resistance profiles of seven isolates collected from the Gulf of Mexico with those of seven clinical strains of P. aeruginosa. Our results indicate that the marine and clinical isolates tested exhibit similar virulence properties; they expressed different virulence factors and were able to kill Galleria mellonella larvae, an animal model commonly used to analyze the pathogenicity of many bacteria, including P. aeruginosa. In contrast, the clinical strains showed higher antibiotic resistance than the marine isolates. Consistently, the clinical strains exhibited a higher prevalence of class 1 integron, an indicator of anthropogenic impact, compared with the marine isolates. Thus, our results indicate that the P. aeruginosa marine strains analyzed in this study, isolated from the Gulf of Mexico, have similar virulence properties, but lower antibiotic resistance, than those from hospitals.

Detection and Quantification of Mono-Rhamnolipids and Di-Rhamnolipids Produced by Pseudomonas aeruginosa.

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This bacterium produces high levels of biosurfactants known as rhamnolipids (RL), which are molecules with significant biotechnological value but are also associated with virulence traits. In this respect, the detection and quantification of RL may be useful for both biotechnology applications and biomedical research projects. In this article, we demonstrate step-by-step the technique to detect the production of the two forms of RL produced by P. aeruginosa using thin-layer chromatography (TLC): mono-rhamnolipids (mRL), molecules constituted by a dimer of fatty acids (mainly C10-C10) linked to one rhamnose moiety, and di-rhamnolipids (dRL), molecules constituted by a similar fatty acid dimer linked to two rhamnose moieties. Additionally, we present a method to measure the total amount of RL based on the acid hydrolysis of these biosurfactants extracted from a P. aeruginosa culture supernatant and the subsequent detection of the concentration of rhamnose that reacts with orcinol. The combination of both techniques can be used to estimate the approximate concentration of mRL and dRL produced by a specific strain, as exemplified here with the type strains PAO1 (phylogroup 1), PA14 (phylogroup 2), and PA7 (phylogroup 3).

The alternative sigma factor RpoS regulates Pseudomonas aeruginosa quorum sensing response by repressing the pqsABCDE operon and activating vfr.

Pseudomonas aeruginosa is an important opportunistic pathogen. Several of its virulence-related processes, including the synthesis of pyocyanin (PYO) and biofilm formation, are controlled by quorum sensing (QS). It has been shown that the alternative sigma factor RpoS regulates QS through the reduction of lasR and rhlR transcription (encoding QS regulators). However, paradoxically, the absence of RpoS increases PYO production and biofilm development (that are RhlR dependent) by unknown mechanisms. Here, we show that RpoS represses pqsE transcription, which impacts the stability and activity of RhlR. In the absence of RpoS, rhlR transcript levels are reduced but not the RhlR protein concentration, presumably by its stabilization by PqsE, whose expression is increased. We also report that PYO synthesis and the expression of pqsE and phzA1B1C1D1E1F1G1 operon exhibit the same pattern at different RpoS concentrations, suggesting that the RpoS-dependent PYO production is due to its ability to modify PqsE concentration, which in turn modulates the activation of the phzA1 promoter by RhlR. Finally, we demonstrate that RpoS favors the expression of Vfr, which activates the transcription of lasR and rhlR. Our study contributes to the understanding of how RpoS modulates the QS response in P. aeruginosa, exerting both negative and positive regulation.

Heterologous production of rhamnolipids in Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 based on the endogenous production of N-acyl-homoserine lactones.

Rhamnolipids (RL) are biosurfactants naturally produced by the opportunistic pathogen Pseudomonas aeruginosa. Currently, RL are commercialized for various applications and produced by Pseudomonas putida due to the health risks associated with their large-scale production by P. aeruginosa. In this work, we show that RL containing one or two rhamnose moieties (mono-RL or di-RL, respectively) can be produced by the innocuous soil-bacterium Pseudomonas chlororaphis subsp chlororaphis ATCC 9446 at titres up to 66 mg/L (about 86% of the production of P. aeruginosa PAO1 in the same culture conditions). The production of RL depends on the expression of P. aeruginosa PAO1 genes encoding the enzymes RhlA, RhlB and RhlC. These genes were introduced in a plasmid, together with a transcriptional regulator (rhlR) forming part of the same operon, with and without RhlC. We show that the activation of rhlAB by RhlR depends on its interaction with P. chlororaphis endogenous acyl-homoserine lactones, which are synthetized by either PhzI or CsaI autoinducer synthases (producing 3-hydroxy-hexanoyl homoserine lactone, 3OH-C6-HSL, or 3-oxo-hexanoyl homoserine lactone, 3O-C6-HSL, respectively). P. chlororaphis transcriptional regulator couple with 3OH-C6-HSL is the primary activator of gene expression for phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production in this soil bacterium. We show that RhlR coupled with 3OH-C6-HSL or 3O-C6-HSL promotes RL production and increases the production of PCA in P. chlororaphis. However, PhzR/3OH-C6-HSL or CsaR/3O-C6-HSL cannot activate the expression of the rhlAB operon to produce mono-RL. These results reveal a complex regulatory interaction between RhlR and P. chlororaphis quorum-sensing signals and highlight the biotechnology potential of P. chlororaphis ATCC 9446 expressing P. aeruginosa rhlAB-R or rhlAB-R-C for the industrial production of RL.

Pseudomonas aeruginosa strains belonging to phylogroup 3 frequently exhibit an atypical quorum sensing response: the case of MAZ105, a tomato rhizosphere isolate.

Pseudomonas aeruginosa is a widespread γ-proteobacterium and an important opportunistic pathogen. The genetically diverse P. aeruginosa phylogroup 3 strains are characterized by producing the pore-forming ExlA toxin and by their lack of a type III secretion system. However, like all strains of this species, they produce several virulence-associated traits, such as elastase, rhamnolipids and pyocyanin, which are regulated by quorum sensing (QS). The P. aeruginosa QS response comprises three systems (Las, Rhl and Pqs, respectively) that hierarchically regulate these virulence factors. The Pqs QS system is composed of the PqsR transcriptional factor, which, coupled with the alkyl-quinolones HHQ or PQS, activates the transcription of the pqsABCDE operon. The products of the first four genes of this operon produce HHQ, which is then converted to PQS by PqsH, while PqsE forms a complex with RhlR and stabilizes it. In this study we report that mutations affecting the Pqs system are particularly common in phylogroup 3 strains. To better understand QS in phylogroup 3 strains we studied strain MAZ105 isolated from tomato rhizosphere and showed that it contains mutations in the central QS transcriptional regulator, LasR, and in the gene encoding the PqsA enzyme involved in the synthesis of PQS. However, it can still produce QS-regulated virulence factors and is virulent in Galleria mellonella and mildly pathogenic in the mouse abscess/necrosis model; our results show that this may be due to the expression of pqsE from a different PqsR-independent promoter than the pqsA promoter. Our results indicate that using anti-virulence therapy based on targeting the PQS system will not be effective against infections by P. aeruginosa phylogroup 3 strains.

Defining the regulatory mechanisms of sigma factor RpoS degradation in Azotobacter vinelandii and Pseudomonas aeruginosa.

In several Gram-negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB-like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti-sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three-hybrid system. We propose that both RssB and RssC are necessary for the ClpXP-dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.

Unraveling the regulation of pyocyanin synthesis by RsmA through MvaU and RpoS in Pseudomonas aeruginosa ID4365.

Pyocyanin is a phenazine with redox activity produced by Pseudomonas aeruginosa that is harmful to other bacteria and eukaryotic organisms by generating reactive oxygen species. Gene regulation of pyocyanin synthesis has been addressed in the PAO1 and PA14 strains and involves the three-quorum sensing systems Las, Rhl, and Pqs; the regulators RsaL, MvaU, and RpoS, and the posttranscriptional Rsm system, among others. Here, we determined how RsmA regulates pyocyanin synthesis in P. aeruginosa ID4365, an overproducer strain. We found that, in the protease peptone glucose ammonium salts medium, rsmA inactivation increases pyocyanin production compared with the wild-type strains ID4365, PAO, and PA14. We showed that RsmA regulates inversely the expression of both phz operons involved in pyocyanin synthesis; particularly the phz2 operon is positively regulated at the transcriptional level indirectly through MvaU. In addition, we found that the phz1 operon contributes mainly to pyocyanin synthesis and that RsmA negatively regulates phzM and phzS expression. Finally, we showed that translation of the sigma factor RpoS is positively regulated by RsmA, and the expression of rpoS under an independent promoter decreases pyocyanin production in the IDrsmA strain. These results indicate that RsmA regulates not only the genes for pyocyanin production but also their regulators.

Pseudomonas aeruginosa LasR overexpression leads to a RsaL-independent pyocyanin production inhibition in a low phosphate condition.

Several Pseudomonas aeruginosa virulence-related traits like pyocyanin are regulated by an intricate regulatory network called quorum sensing (QS) that relies on transcriptional regulators that are activated through binding to a self-produced molecule called an autoinducer (AI). QS is composed of three systems, Las, Rhl and Pqs. In the Las system, the regulatory protein LasR interacts with its AI to activate the other two QS systems. In turn, the Rhl and Pqs systems regulate the expression of multiple virulence-related genes, such as the genes of the reiterated operons phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2 involved in pyocyanin production. The Las system also regulates the negative regulator RsaL, which provides negative feedback to the QS-response, including repression of pyocyanin synthesis genes. In this work, we describe that LasR can act as a negative regulator of phzA1 transcription and hence of pyocyanin production and that this regulation is independent of RsaL activity. This work contributes to the understanding of QS-dependent pyocyanin production and demonstrates a previously uncharacterized role of LasR as a repressor.

Overview on Glycosylated Lipids Produced by Bacteria and Fungi: Rhamno-, Sophoro-, Mannosylerythritol and Cellobiose Lipids.

Wide ranges of microorganisms produce glycosylated lipids (GL), which are characterized by their tensio-active properties. Therefore, they can be used in different industrial applications as biosurfactants, such as food, agriculture, cosmetics, and health products among others. Two GL biosurfactants, rhamnolipids (RL) and sophorolipids (SL), are now commercially available and share a significant part of the biosurfactant market that in 2017 represented about 2.5% of the total surfactants market, estimated at 15 million tons globally.In this chapter, we present a general overview of GL biosurfactants in terms of their diversity and the microorganisms that produce them. Additionally, we focus on the more detailed description of RL, SL, mannosylerythritol lipids (MEL), and cellobiose lipids (CL).Pseudomonas aeruginosa, the ubiquitous opportunistic pathogenic bacterium, is the best RL producer, but other non-pathogenic bacteria like Burkholderia thailandensis and Pseudomonas chlororaphis NRRL B-30761 are also capable of producing them naturally. In addition, Pseudomonas putida has been used as heterologous host to produce RL with good yields. Here we describe the biosynthetic pathway for RL production, the genes involved in its synthesis, and some of the challenges for producing a homogeneous RL product in high quantities that is suitable for specific applications.SL, MEL, and CL are some of the GL biosurfactants produced in high quantities by fungi, like Starmerella bombicola, Moesziomyces aphidis, or Ustilago maydis. We provide an overview of some of their characteristics, insights on the metabolic pathways involved in their synthesis and genetic modifications performed to increase their production, as well as fermentation and purification strategies and some of their applications.

PqsR-independent quorum-sensing response of Pseudomonas aeruginosa ATCC 9027 outlier-strain reveals new insights on the PqsE effect on RhlR activity.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen that represents an important health hazard. The quorum-sensing response regulates the expression of several virulence factors and involves three regulons: Las, Rhl, and Pqs. The P. aeruginosa ATCC 9027 strain, which belongs to the genetically diverse PA7 clade, contains a frame-shift mutation in the pqsR gene that encodes a transcriptional activator necessary for pyocyanin (PYO) synthesis in type strains PAO1 and PA14. Here we characterize the PqsE-dependent production of PYO in strain ATCC 9027. We show that this strain expresses pqsE independently of PqsR and in the absence of quinolone production, and that PqsE promotes the RhlR-dependent production of PYO, yet this production is not strictly dependent on PqsE. In addition, we show that in both strains ATCC 9027 and PAO1, PqsE overexpression causes an increased concentration of RhlR and enhances PYO production but does not affect rhamnolipids (RL) production in the same way. These results suggest that PqsE interaction with RhlR preferentially modifies its ability to activate transcription of genes involved in PYO production and provide new evidence about PqsE-dependent RhlR activation, highlighting the variability of the QS response among different P. aeruginosa clades and strains. HIGHLIGHTS: Pseudomonas aeruginosa ATCC 9027 is able to produce pyocyanin in phosphate limiting conditions, even in the absence of a functional PqsR. This strain does not produce alkyl quinolones like PQS and HHQ, but expresses pqsE. Synthesis of pyocyanin by ATCC 9027 is only partially dependent on pqsE. The overexpression of pqsE in the ATCC 9027 and PAO1 strains causes pyocyanin overproduction. The overexpression of pqsE in these strains causes an increased RhlR concentration without affecting rhlR transcription or translation. Rhamnolipids production is not affected to the same extent as pyocyanin by overexpression of pqsE in these strains.

Vfr or CyaB promote the expression of the pore-forming toxin exlBA operon in Pseudomonas aeruginosa ATCC 9027 without increasing its virulence in mice.

Pseudomonas aeruginosa is a wide-spread γ-proteobacterium that produces the biosurfactant rhamnolipid that has a great commercial value due to excellent properties of low toxicity and high biodegradability. However, this bacterium is an opportunist pathogen that constitutes an important health hazard due to its production of virulence-associated traits and its high antibiotic resistance. Thus, it is highly desirable to have a non-virulent P. aeruginosa strain for rhamnolipid production. It has been reported that strain ATCC 9027 is avirulent in mouse models of infection, and it is still able to produce rhamnolipid. Thus, it has been proposed to be suitable for it industrial production, since it encodes a defective LasR quorum sensing (QS) transcriptional regulator that is the head of this regulatory network. However, the restoration of virulence factor production by overexpression of rhlR (the gene encoding a QS-transcriptional regulator which is under the transcriptional control of LasR) is not sufficient to restore its virulence in mice. It is desirable to obtain a deeper understanding of ATCC 9027 attenuated-virulence phenotype and to assess the safety of this strain to be used at an industrial scale. In this work we determined whether increasing the expression of the pore-forming toxin encoded by the exlBA operon in strain ATCC 9027 had an impact on its virulence using Galleria mellonella and mouse models of infections. We increased the expression of the exlBA operon by overexpressing from a plasmid its transcriptional activator Vfr or of the Vfr ligand cyclic AMP produced by CyaB. We found that in G. mellonella ATCC 9027/pUCP24-vfr and ATCC 9027/pUCP24-cyaB gained a virulent phenotype, but these strains remained avirulent in murine models of P. aeruginosa infection. These results reinforce the possibility of using ATCC 9027 for industrial biosurfactants production.

The Rhl Quorum-Sensing System Is at the Top of the Regulatory Hierarchy under Phosphate-Limiting Conditions in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is a major nosocomial pathogen that presents high-level resistance to antibiotics. Its ability to cause infections relies on the production of multiple virulence factors. Quorum sensing (QS) regulates the expression of many of these virulence factors through three QS systems: Las, Rhl, and PQS. The Las system positively regulates the other two systems, so it is at the top of a hierarchized regulation. Nevertheless, clinical and environmental strains that lack a functional Las system have been isolated, and, surprisingly, some of them still have the ability to produce virulence factors and infect animal models, so it has been suggested that the hierarchy is flexible under some conditions or with atypical strains. Here, we analyze the PAO1 type strain and its ΔlasR-derived mutant and report, for the first time, a growth condition (phosphate limitation) where LasR absence has no effect either on virulence factor production or on the gene expression profile, in contrast to a condition of phosphate repletion where the LasR hierarchy is maintained. This work provides evidence on how the QS hierarchy can change from being a strictly LasR-dependent to a LasR-independent RhlR-based hierarchy under phosphate limitation even in the PAO1 type strain.IMPORTANCEPseudomonas aeruginosa is an important pathogen, considered a priority for the development of new therapeutic strategies. An important approach to fight its infections relies on blocking quorum sensing. The Las system is the main regulator of the quorum-sensing response, so many research efforts aim to block this system to suppress the entire response. In this work, we show that LasR is dispensable in a phosphate-limited environment in the PAO1 type strain, which has been used to define the quorum-sensing response hierarchy, and that under this condition RhlR is at the top of the regulation hierarchy. These results are highly significant, since phosphate limitation represents a similar environment to the one that P. aeruginosa faces when establishing infections.

Rhamnolipids produced by Pseudomonas: from molecular genetics to the market.

Rhamnolipids are biosurfactants with a wide range of industrial applications that entered into the market a decade ago. They are naturally produced by Pseudomonas aeruginosa and some Burkholderia species. Occasionally, some strains of different bacterial species, like Pseudomonas chlororaphis NRRL B-30761, which have acquired RL-producing ability by horizontal gene transfer, have been described. P. aeruginosa, the ubiquitous opportunistic pathogenic bacterium, is the best rhamnolipids producer, but Pseudomonas putida has been used as heterologous host for the production of this biosurfactant with relatively good yields. The molecular genetics of rhamnolipids production by P. aeruginosa has been widely studied not only due to the interest in developing overproducing strains, but because it is coordinately regulated with the expression of different virulence-related traits by the quorum-sensing response. Here, we highlight how the research of the molecular mechanisms involved in rhamnolipid production have impacted the development of strains that are suitable for industrial production of this biosurfactant, as well as some perspectives to improve these industrial useful strains.

Tracking the genome of four Pseudomonas aeruginosa isolates that have a defective Las quorum-sensing system, but are still virulent.

In this work we analysed the whole genome extended multilocus sequence typing (wgMLST) of four Pseudomonas aeruginosa strains that are characterized by being virulent despite having a defective Las quorum-sensing (QS) system, and compare them with the wgMLST of the PAO1 and PA14 type strains. This comparison was done to determine whether there was a genomic characteristic that was common to the strains with an atypical QS response. The analysed strains include two environmental isolates (ID 4365 isolated from the Indian Ocean, and M66 isolated from the Churince water system in Cuatro Ciénegas Coahuila, México), one veterinary isolate (strain 148 isolated from the stomach of a dolphin) and a clinical strain (INP43 that is a cystic fibrosis pediatric isolate). We determine that the six analysed strains have a core genome of 4689 loci that was used to construct a wgMLST-phylogeny tree. Using the cano-wgMLST_BacCompare software we found that there was no common genomic characteristic to the strains with an atypical QS-response and we identify ten loci that are highly discriminatory of the six strains' phylogeny so that their MLST can reconstruct the wgMLST-phylogeny tree of these strains. We discuss here the nature of these ten highly discriminatory genes in the context of P. aeruginosa virulence and evolution.

Virulence factors regulation by the quorum-sensing and Rsm systems in the marine strain Pseudomonas aeruginosa ID4365, a natural mutant in lasR.

Pseudomonas aeruginosa is an opportunistic human pathogen that is able to produce several virulence factors such as pyocyanin, rhamnolipids and elastase. In the clinical reference strain PAO1, synthesis of these virulence factors is regulated transcriptionally by quorum sensing (QS) and post-transcriptionally by the Rsm system. Herein, we investigated the role of these systems in the control of the pyocyanin, rhamnolipids and elastase production in the marine strain ID4365. We found that this strain carries a nonsense mutation in lasR that makes it a natural mutant in the Las QS system. However, its QS response is still functional with the Rhl system activating virulence factors synthesis. We found that the Rsm system affects virulence factors production, since overexpression of RsmA reduces pyocyanin production whereas RsmY overexpression increases its synthesis. Unexpectedly, and in contrast to the type strain PAO1, inactivation of rsmA increases pyocyanin but reduces elastase and rhamnolipids production by a reduction of RhlR levels. Thus, QS and Rsm systems are involved in regulating virulence factors production, but this regulation is different to the PAO1 strain even though their genomes are highly conserved. It is likely that these differences are related to the different ecological niches in which these strains lived.

Rhamnolipids stabilize quorum sensing mediated cooperation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the main models to study social behaviors in bacteria since it synthesizes several exoproducts, including exoproteases and siderophores and release them to the environment. Exoproteases and siderophores are public goods that can be utilized by the individuals that produce them but also by non-producers, that are considered social cheaters. Molecularly exoprotease cheaters are mutants in regulatory genes such as lasR, and are commonly isolated from chronic infections and selected in the laboratory upon serial cultivation in media with protein as a sole carbon source. Despite that the production of exoproteases is exploitable, cooperators have also ways to restrict the growth and selection of social cheaters, for instance by producing toxic metabolites like pyocyanin. In this work, using bacterial competitions, serial cultivation and growth assays, we demonstrated that rhamnolipids which production is regulated by quorum sensing, selectively affect the growth of lasR mutants and are able to restrict social cheating, hence contributing to the maintenance of cooperation in Pseudomonas aeruginosa populations.

The third quorum-sensing system of Pseudomonas aeruginosa: Pseudomonas quinolone signal and the enigmatic PqsE protein.

Pseudomonas aeruginosa is an opportunistic pathogen that produces several virulence factors such as lectin A, pyocyanin, elastase and rhamnolipids. These compounds are controlled transcriptionally by three quorum-sensing circuits, two based on the synthesis and detection of N-acyl-homoserine-lactone termed the Las and Rhl system and a third system named the Pseudomonas quinolone signal (PQS) system, which is responsible for generating 2-alkyl-4(1 h)-quinolones (AQs). The transcriptional regulator called PqsR binds to the promoter of pqsABCDE in the presence of PQS or HHQ creating a positive feedback-loop. PqsE, encoded in the operon for AQ synthesis, is a crucial protein for pyocyanin production, activating the Rhl system by a still not fully understood mechanism. In turn, the regulation of the PQS system is modulated by Las and Rhl systems, which act positively and negatively, respectively. This review focuses on the PQS system, from its discovery to its role in Pseudomonas pathogenesis, such as iron depletion and pyocyanin synthesis that involves the PqsE protein - an intriguing player of this system.