The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidated. Key components of the pqs QS system are 2-heptyl-4-hydroxyquinoline (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), the transcriptional regulator PqsR and the PQS-effector element PqsE. To define the individual contribution of each of these components to QS-mediated regulation, transcriptomic analyses were performed and validated on engineered P. aeruginosa strains in which the biosynthesis of 2-alkyl-4-quinolones (AQs) and expression of pqsE and pqsR have been uncoupled, facilitating the identification of the genes controlled by individual pqs system components. The results obtained demonstrate that i) the PQS biosynthetic precursor HHQ triggers a PqsR-dependent positive feedback loop that leads to the increased expression of only the pqsABCDE operon, ii) PqsE is involved in the regulation of diverse genes coding for key virulence determinants and biofilm development, iii) PQS promotes AQ biosynthesis, the expression of genes involved in the iron-starvation response and virulence factor production via PqsR-dependent and PqsR-independent pathways, and iv) HQNO does not influence transcription and hence does not function as a QS signal molecule. Overall this work has facilitated identification of the specific regulons controlled by individual pqs system components and uncovered the ability of PQS to contribute to gene regulation independent of both its ability to activate PqsR and to induce the iron-starvation response. Many bacterial pathogens control virulence gene expression and the development of antibiotic-resistant biofilms via intercellular communication through ‘quorum sensing’ (QS). QS systems depend on the synthesis, secretion and perception of diffusible signalling molecules that enable bacteria to synchronize their behaviour at the population level and are considered ideal targets for the development of anti-virulence drugs. Pseudomonas aeruginosa employs several overlapping QS circuits including the pqs system to control the expression of virulence determinants. The pqs QS system relies on multiple 2-alkyl-4-quinolones (AQs), including the Pseudomonas Quinolone Signal (PQS), as signal molecules. However, the individual contributions of key AQs and the effector proteins PqsR and PqsE within the auto-regulated pqs system have not been elucidated because of their inter-dependence. By constructing P. aeruginosa strains with multiple mutations in the pqs system and determining their transcriptomes in the presence or absence of PqsR, PqsE or exogenously supplied AQs, we define the distinct regulons involved and characterize a novel PQS signalling pathway independent of PqsR and the iron-starvation response.

The major human bacterial pathogenPseudomonas aeruginosacauses multidrug-resistant infections in people with underlying immunodeficiencies or structural lung diseases such as cystic fibrosis (CF). We show that a few environmental isolates, driven by horizontal gene acquisition, have become dominant epidemic clones that have sequentially emerged and spread through global transmission networks over the past 200 years. These clones demonstrate varying intrinsic propensities for infecting CF or non-CF individuals (linked to specific transcriptional changes enabling survival within macrophages); have undergone multiple rounds of convergent, host-specific adaptation; and have eventually lost their ability to transmit between different patient groups. Our findings thus explain the pathogenic evolution ofP. aeruginosaand highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.

The long-term use of antibiotics has led to the emergence of multi-drug-resistant bacteria. A promising strategy to combat bacterial infections aims at hampering their adaptability to the host environment without affecting growth. In this context, the intercellular communication system quorum sensing (QS), which controls virulence factor production and biofilm formation in diverse human pathogens, is considered an ideal target. Here we describe the identification of new inhibitors of the pqs QS system of the human pathogen Pseudomonas aeruginosa, by screening a library of 1,600 FDA-approved drugs. Phenotypic characterization of ad hoc engineered strains and in silico molecular docking demonstrated that the antifungal drugs clotrimazole and miconazole, and an antibacterial compound active against Gram-positive pathogens, clofoctol, inhibit the pqs system, probably by targeting the transcriptional regulator PqsR. The most active inhibitor, clofoctol, specifically inhibited the expression of pqs-controlled virulence traits in P. aeruginosa, such as pyocyanin production, swarming motility, biofilm formation, and expression of genes involved in siderophore production. Moreover, clofoctol protected Galleria mellonella larvae from P. aeruginosa infection and inhibited the pqs QS system in P. aeruginosa isolates from cystic fibrosis patients. Notably, clofoctol is already approved for clinical treatment of pulmonary infections caused by Gram-positive bacterial pathogens, hence this drug has considerable clinical potential as an antivirulence agent for the treatment of P. aeruginosa lung infections.

Recent developments in bottom-up synthetic biology (e.g., lipid vesicle technology integrated with cell-free protein expression systems) allow the generation of semi-synthetic minimal cells (in short, synthetic cells, SCs) endowed with some distinctive capacities of natural cells. In particular, such approaches provide technological tools and conceptual frameworks for the design and engineering of programmable SCs capable of communicating with natural cells by exchanging chemical signals. Here we describe the generation of giant vesicle-based SCs which, via gene expression, synthesize in their aqueous lumen an enzyme that in turn produces a chemical signal. The latter is a small molecule, which is passively released in the medium and then perceived by the bacterium Pseudomonas aeruginosa, demonstrating that SCs and bacteria can communicate chemically. The results pave the way to a novel basic and applied research area where synthetic cells can communicate with natural cells, for example for exploring minimal cognition, developing chemical information technologies, and producing smart and programmable drug-producing/drug-delivery systems.

Acinetobacter baumannii is an important nosocomial pathogen. Mechanisms that allow A. baumannii to cause human infection are still poorly understood. Iron is an essential nutrient for bacterial growth in vivo, and the multiplicity of iron uptake systems in A. baumannii suggests that iron acquisition contributes to the ability of A. baumannii to cause infection. In Gram-negative bacteria, active transport of ferrisiderophores and heme relies on the conserved TonB-ExbB-ExbD energy-transducing complex, while active uptake of ferrous iron is mediated by the Feo system. The A. baumannii genome invariably contains three tonB genes (tonB1, tonB2, and tonB3), whose role in iron uptake is poorly understood. Here, we generated A. baumannii mutants with knockout mutations in the feo and/or tonB gene. We report that tonB3 is essential for A. baumannii growth under iron-limiting conditions, whereas tonB1, tonB2, and feoB appear to be dispensable for ferric iron uptake. tonB3 deletion resulted in reduced intracellular iron content despite siderophore overproduction, supporting a key role of TonB3 in iron uptake. In contrast to the case for tonB1 and tonB2, the promoters of tonB3 and feo contain functional Fur boxes and are upregulated in iron-poor media. Both TonB3 and Feo systems are required for growth in complement-free human serum and contribute to resistance to the bactericidal activity of normal human serum, but only TonB3 appears to be essential for virulence in insect and mouse models of infection. Our findings highlight a central role of the TonB3 system for A. baumannii pathogenicity. Hence, TonB3 represents a promising target for novel antibacterial therapies and for the generation of attenuated vaccine strains.

The ability of Pseudomonas aeruginosa to cause difficult-to-treat infections relies on its capacity to fine-tune the expression of multiple virulence traits via the las , rhl , and pqs QS systems. Both the pqs effector protein PqsE and the rhl transcriptional regulator RhlR are required for full production of key virulence factors in vitro and pathogenicity in vivo .