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Pseudomonas aeruginosa is a versatile bacterium found in various environments. It can cause severe infections in immunocompromised patients and naturally resists many antibiotics. The World Health Organization listed it among the top priority pathogens for research and development of new antimicrobial compounds. Quorum sensing (QS) is a cell-cell communication mechanism, which is important for P. aeruginosa adaptation and pathogenesis. Here, we validate the central role of the PqsE protein in QS particularly by its impact on the regulator RhlR. This study challenges the traditional dogmas of QS regulation in P. aeruginosa and ties loose ends in our understanding of the traditional QS circuit by confirming RhlR to be the main QS regulator in P. aeruginosa . PqsE could represent an ideal target for the development of new control methods against the virulence of P. aeruginosa . This is especially important when considering that LasR-defective mutants frequently arise, e.g., in chronic infections. The bacterium Pseudomonas aeruginosa has emerged as a central threat in health care settings and can cause a large variety of infections. It expresses an arsenal of virulence factors and a diversity of survival functions, many of which are finely and tightly regulated by an intricate circuitry of three quorum sensing (QS) systems. The las system is considered at the top of the QS hierarchy and activates the rhl and pqs systems. It is composed of the LasR transcriptional regulator and the LasI autoinducer synthase, which produces 3-oxo-C 12 -homoserine lactone (3-oxo-C 12 -HSL), the ligand of LasR. RhlR is the transcriptional regulator for the rhl system and is associated with RhlI, which produces its cognate autoinducer C 4 -HSL. The third QS system is composed of the pqsABCDE operon and the MvfR (PqsR) regulator. PqsABCD synthetize 4-hydroxy-2-alkylquinolines (HAQs), which include ligands activating MvfR. PqsE is not required for HAQ production and instead is associated with the expression of genes controlled by the rhl system. While RhlR is often considered the main regulator of rhlI , we confirmed that LasR is in fact the principal regulator of C 4 -HSL production and that RhlR regulates rhlI and production of C 4 -HSL essentially only in the absence of LasR by using liquid chromatography-mass spectrometry quantifications and gene expression reporters. Investigating the expression of RhlR targets also clarified that activation of RhlR-dependent QS relies on PqsE, especially when LasR is not functional. This work positions RhlR as the key QS regulator and points to PqsE as an essential effector for full activation of this regulation. IMPORTANCE Pseudomonas aeruginosa is a versatile bacterium found in various environments. It can cause severe infections in immunocompromised patients and naturally resists many antibiotics. The World Health Organization listed it among the top priority pathogens for research and development of new antimicrobial compounds. Quorum sensing (QS) is a cell-cell communication mechanism, which is important for P. aeruginosa adaptation and pathogenesis. Here, we validate the central role of the PqsE protein in QS particularly by its impact on the regulator RhlR. This study challenges the traditional dogmas of QS regulation in P. aeruginosa and ties loose ends in our understanding of the traditional QS circuit by confirming RhlR to be the main QS regulator in P. aeruginosa . PqsE could represent an ideal target for the development of new control methods against the virulence of P. aeruginosa . This is especially important when considering that LasR-defective mutants frequently arise, e.g., in chronic infections.

Successful colonization of a multitude of ecological niches by the bacterium Pseudomonas aeruginosa relies on its ability to respond to concentrations of self-produced signal molecules. This intercellular communication system known as quorum sensing (QS) tightly regulates the expression of virulence determinants and a diversity of survival functions, including those required for social behaviors. In planktonic cultures of P. aeruginosa, the transcriptional regulator LasR is generally considered on top of the QS circuitry hierarchy; its activation relies on binding to 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), a product of LasI synthase. Transcription of lasI is activated by LasR, resulting in a positive feedback loop. Few studies have looked at the function of QS during surface growth even though P. aeruginosa typically lives in biofilm-like communities under natural conditions. Here, we show that surface-grown P. aeruginosa, including prototypical strain PA14, produces 3-oxo-C12-HSL in the absence of LasR. This phenotype is commonly observed upon surface association in naturally occurring environmental and clinical LasR-defective isolates, suggesting a conserved alternative function for the signal. Notably, in surface-grown cultures, 3-oxo-C12-HSL reaches higher levels than planktonic cells, and there is a delayed timing of its production. Accordingly, 3-oxo-C12-HSL upregulates the autologous expression of pyocyanin and LasR-controlled virulence determinants in neighboring cells even in the absence of the cognate regulator LasR. This highlights a possible role for 3-oxo-C12-HSL in shaping community responses and provides a possible evolutive benefit for mixed populations to carry LasR-defective cells, a common feature of natural populations of P. aeruginosa. IMPORTANCE The bacterium Pseudomonas aeruginosa colonizes and thrives in many environments, in which it is typically found in surface-associated polymicrobial communities known as biofilms. Adaptation to this social behavior is aided by quorum sensing (QS), an intercellular communication system pivotal in the expression of social traits. Regardless of its importance in QS regulation, the loss of function of the master regulator LasR is now considered a conserved adaptation of P. aeruginosa, irrespective of the origin of the strains. By investigating the QS circuitry in surface-grown cells, we found an accumulation of QS signal 3-oxo-C12-HSL in the absence of its cognate receptor and activator, LasR. The current understanding of the QS circuit, mostly based on planktonic growing cells, is challenged by investigating the QS circuitry of surface-grown cells. This provides a new perspective on the beneficial aspects that underline the frequency of LasR-deficient isolates.

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa ( PA ) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti- PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA -driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections. Pseudomonas aeruginosa infections are increasingly difficult to treat due to the development of antimicrobial resistance. Here, the authors describe the synthesis, characterisation and efficacy of a quorum sensing inhibitor.

c-di-GMP is a major player in the switch between biofilm and motile lifestyles. Several bacteria exhibit a large number of c-di-GMP metabolizing proteins, thus a fine-tuning of this nucleotide levels may occur. It is hypothesized that some c-di-GMP metabolizing proteins would provide the global c-di-GMP levels inside the cell whereas others would maintain a localized pool, with the resulting c-di-GMP acting at the vicinity of its production. Although attractive, this hypothesis has yet to be demonstrated in Pseudomonas aeruginosa . We found that the diguanylate cyclase DgcP interacts with the cytosolic region of FimV, a polar peptidoglycan-binding protein involved in type IV pilus assembly. Moreover, DgcP is located at the cell poles in wild type cells but scattered in the cytoplasm of cells lacking FimV. Overexpression of dgcP leads to the classical phenotypes of high c-di-GMP levels (increased biofilm and impaired motilities) in the wild-type strain, but not in a Δ fimV background. Therefore, our findings suggest that DgcP activity is regulated by FimV. The polar localization of DgcP might contribute to a local c-di-GMP pool that can be sensed by other proteins at the cell pole, bringing to light a specialized function for a specific diguanylate cyclase.

, a bacterium causing infections in immunocompromised individuals, regulates several of its virulence functions using three interlinked quorum sensing (QS) systems ( , , and ). Despite its presumed importance in regulating virulence, dysfunction of the system regulator LasR occurs frequently in strains isolated from various environments, including clinical infections. This newfound abundance of LasR-defective strains calls into question existing hypotheses regarding their selection. Indeed, current assumptions concerning factors driving the emergence of LasR-deficient isolates and the role of LasR in the QS hierarchy must be reconsidered. Here, we propose that LasR is not the primary master regulator of QS in all genetic backgrounds, even though it remains ecologically significant. We also revisit and complement current knowledge on the ecology of LasR-dependent QS in , discuss the hypotheses explaining the putative adaptive benefits of selecting against LasR function, and consider the implications of this renewed understanding.

The bacterium Pseudomonas aeruginosa has emerged as a central threat in health care settings and can cause a large variety of infections. It expresses an arsenal of virulence factors and a diversity of survival functions, many of which are finely and tightly regulated by an intricate circuitry of three quorum sensing (QS) systems. The las system is considered at the top of the QS hierarchy and activates the rhl and pqs systems. It is composed of the LasR transcriptional regulator and the LasI autoinducer synthase, which produces 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), the ligand of LasR. RhlR is the transcriptional regulator for the rhl system and is associated with RhlI, which produces its cognate autoinducer C4-HSL. The third QS system is composed of the pqsABCDE operon and the MvfR (PqsR) regulator. PqsABCD synthetize 4-hydroxy-2-alkylquinolines (HAQs), which include ligands activating MvfR. PqsE is not required for HAQ production and instead is associated with the expression of genes controlled by the rhl system. While RhlR is often considered the main regulator of rhlI, we confirmed that LasR is in fact the principal regulator of C4-HSL production and that RhlR regulates rhlI and production of C4-HSL essentially only in the absence of LasR by using liquid chromatography-mass spectrometry quantifications and gene expression reporters. Investigating the expression of RhlR targets also clarified that activation of RhlR-dependent QS relies on PqsE, especially when LasR is not functional. This work positions RhlR as the key QS regulator and points to PqsE as an essential effector for full activation of this regulation. IMPORTANCE Pseudomonas aeruginosa is a versatile bacterium found in various environments. It can cause severe infections in immunocompromised patients and naturally resists many antibiotics. The World Health Organization listed it among the top priority pathogens for research and development of new antimicrobial compounds. Quorum sensing (QS) is a cell-cell communication mechanism, which is important for P. aeruginosa adaptation and pathogenesis. Here, we validate the central role of the PqsE protein in QS particularly by its impact on the regulator RhlR. This study challenges the traditional dogmas of QS regulation in P. aeruginosa and ties loose ends in our understanding of the traditional QS circuit by confirming RhlR to be the main QS regulator in P. aeruginosa. PqsE could represent an ideal target for the development of new control methods against the virulence of P. aeruginosa. This is especially important when considering that LasR-defective mutants frequently arise, e.g., in chronic infections.

Successful colonization of a multitude of ecological niches by the bacterium Pseudomonas aeruginosa relies on its ability to respond to concentrations of self-produced signal molecules. This intercellular communication system known as quorum sensing (QS) tightly regulates the expression of virulence determinants and a diversity of survival functions, including those required for social behaviors. In planktonic cultures of P. aeruginosa , the transcriptional regulator LasR is generally considered on top of the QS circuitry hierarchy; its activation relies on binding to 3-oxo-C 12 -homoserine lactone (3-oxo-C 12 -HSL), a product of LasI synthase. Transcription of lasI is activated by LasR, resulting in a positive feedback loop. Few studies have looked at the function of QS during surface growth even though P. aeruginosa typically lives in biofilm-like communities under natural conditions. Here, we show that surface-grown P. aeruginosa , including prototypical strain PA14, produces 3-oxo-C 12 -HSL in the absence of LasR. This phenotype is commonly observed upon surface association in naturally occurring environmental and clinical LasR-defective isolates, suggesting a conserved alternative function for the signal. Notably, in surface-grown cultures, 3-oxo-C12-HSL reaches higher levels than planktonic cells, and there is a delayed timing of its production. Accordingly, 3-oxo-C 12 -HSL upregulates the autologous expression of pyocyanin and LasR-controlled virulence determinants in neighboring cells even in the absence of the cognate regulator LasR. This highlights a possible role for 3-oxo-C 12 -HSL in shaping community responses and provides a possible evolutive benefit for mixed populations to carry LasR-defective cells, a common feature of natural populations of P. aeruginosa . The bacterium Pseudomonas aeruginosa colonizes and thrives in many environments, in which it is typically found in surface-associated polymicrobial communities known as biofilms. Adaptation to this social behavior is aided by quorum sensing (QS), an intercellular communication system pivotal in the expression of social traits. Regardless of its importance in QS regulation, the loss of function of the master regulator LasR is now considered a conserved adaptation of P. aeruginosa , irrespective of the origin of the strains. By investigating the QS circuitry in surface-grown cells, we found an accumulation of QS signal 3-oxo-C 12 -HSL in the absence of its cognate receptor and activator, LasR. The current understanding of the QS circuit, mostly based on planktonic growing cells, is challenged by investigating the QS circuitry of surface-grown cells. This provides a new perspective on the beneficial aspects that underline the frequency of LasR-deficient isolates.

Opportunistic bacteria, as adaptable microorganisms, continually adjust to dynamic environments by fine-tuning gene expression at the transcriptional level. While transitioning from a saprophytic lifestyle to human infection, bacteria confront challenges like acclimating to elevated temperatures and securing attachment to host tissues for proliferation. A critical aspect of bacterial adaptation involves sensing fluctuations in cellular density, governed by quorum sensing (QS), an intercellular communication system. Within QS, bacteria produce signalling molecules whose concentrations increase with cellular density. Upon reaching a critical threshold, indicated by a specific concentration of these molecules, QS activates. This activation triggers transcriptional regulators, orchestrating coordinated responses essential for bacterial virulence. Given the rising threat of antibiotic resistance, there is growing interest in targeting QS to attenuate bacterial virulence. However, the success of this strategy hinges on a fundamental comprehension of QS function in bacteria. One of the most well-studied QS systems is from Pseudomonas aeruginosa, offering valuable insights into bacterial pathogenicity and potential therapeutic targets. P. aeruginosaQS comprises three intertwined systems: the las, rhl, and pqs. Each system has one transcriptional regulator (LasR, RhlR, and MvfR) and one main cognate autoinducer. Although QS function is interconnected, LasR-defective isolates persist within P. aeruginosa populations across clinical and non-clinical environments. The ecological relevance of these LasR-defective isolates was explored in the presented thesis, integrating their presence with often encountered environmental cues, namely surface sensing and temperature variations. The first research chapter delves into QS functions of surface-grown cells, mostly addressing the production of the cognate signal molecule from the las system, known as N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). While dependent on the presence of LasR in its production in planktonic cells, this requirement is absent in surface-associated conditions. The second half of this document focuses on temperature variations and QS function. In LasR-defective isolates, RhlR is the regulator that sustains group responses in these isolates. RhlR function depends on the presence of QS-produced elements that stabilize it. Akin to these factors, a lower temperature also induces RhlR activity. Environmental-like temperature also impacts the QS function of LasRdefective isolates, exemplified by the characterization of the naturally evolved LasR A158P. Understanding these environmental-driven QS nuances is pivotal in deciphering the evolutionary trajectories of P. aeruginosa and guiding targeted therapeutic interventions.

Successful colonization of a multitude of ecological niches by the bacterium Pseudomonas aeruginosa relies on its ability to respond to concentrations of self-produced signal molecules. This intercellular communication system known as quorum sensing (QS) tightly regulates the expression of virulence determinants and a diversity of survival functions, including those required for social behaviours. In planktonic cultures of P. aeruginosa, the transcriptional regulator LasR is generally considered on top of the QS circuitry hierarchy; its activation relies on binding to 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), a product of the LasI synthase. Transcription of lasI is activated by LasR, resulting in a positive feedback loop. Few studies have looked at the function of QS during surface growth even though P. aeruginosa typically lives in biofilm-like communities under natural conditions. Here, we show that surface-grown P. aeruginosa readily produces 3-oxo-C12-HSL in absence of LasR, and that this phenotype is frequent upon surface association in naturally occurring environmental and clinical LasR-defective isolates, suggesting a conserved alternative function for the signal. Indeed, even in the absence of the cognate regulator LasR, 3-oxo-C12-HSL upregulates the autologous expression of pyocyanin and of LasR-controlled virulence determinants in neighboring cells. This highlights a possible role for 3-oxo-C12-HSL in shaping community responses and provides a possible evolutive benefit for mixed populations to carry LasR-defective cells, a common feature of natural of P. aeruginosa. The bacterium Pseudomonas aeruginosa colonizes and thrives in many environments, in which it is typically found in surface-associated polymicrobial communities known as biofilms. Adaptation to this social behavior is aided by quorum sensing (QS), an intercellular communication system pivotal in the expression of social traits. Regardless of its importance in QS regulation, the loss of function of the master regulator LasR is now considered a conserved adaptation of P. aeruginosa, irrespective of the origin of strains. By investigating the QS circuitry in surface-grown cells, we found accumulation of QS signal 3-oxo-C12-HSL in absence of its cognate receptor and activator, LasR. The current understanding of the QS circuit, mostly based on planktonic growing cells, is challenged by investigating the QS circuitry of surface-grown cells. This provides a new perspective on the beneficial aspects that underline the frequency of LasR-deficient isolates.