Evolution and host-specific adaptation of Pseudomonas aeruginosa.

The major human bacterial pathogen Pseudomonas aeruginosa causes 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 of P. aeruginosa and highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.

Hydrogen sulfide production does not affect antibiotic resistance in Pseudomonas aeruginosa.

Hydrogen sulfide (H2S) has been proposed to protect bacteria from antibiotics, pointing to H2S-producing enzymes as possible targets for the development of antibiotic adjuvants. Here, MIC assays performed with Pseudomonas aeruginosa mutants producing altered H2S levels demonstrate that H2S does not affect antibiotic resistance in this bacterium. Moreover, correlation analyses in a large collection of P. aeruginosa cystic fibrosis isolates argue against the protective role of H2S from antibiotic activity during chronic lung infection.

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa.

The outer membrane (OM) is an essential structure of Gram-negative bacteria that provides mechanical strength and protection from large and/or hydrophobic toxic molecules, including many antibiotics. The OM is composed of glycerophospholipids (GPLs) and lipopolysaccharide (LPS) in the inner and outer leaflets, respectively, and hosts integral ß-barrel proteins and lipoproteins. While the systems responsible for translocation and insertion of LPS and OM proteins have been elucidated, the mechanism(s) mediating transport of GPLs from the inner membrane to the OM has remained elusive for decades. Very recently, studies performed in Escherichia coli proposed a role in this process for AsmA-like proteins that are predicted to share structural features with eukaryotic lipid transporters. In this study, we provide the first systematic investigation of AsmA-like proteins in a bacterium other than E. coli, the opportunistic human pathogen Pseudomonas aeruginosa. Bioinformatic analyses revealed that P. aeruginosa possesses seven AsmA-like proteins. Deletion of asmA-like genes in many different combinations, coupled with conditional mutagenesis, revealed that four AsmA-like proteins are redundantly essential for growth and OM integrity in P. aeruginosa, including a novel AsmA-like protein (PA4735) that is not present in E. coli. Cells depleted of AsmA-like proteins showed severe defects in the OM permeability barrier that were partially rescued by lowering the synthesis or transport of LPS. Since fine balancing of GPL and LPS levels is crucial for OM integrity, this evidence supports the role of AsmA-like proteins in GPL transport toward the OM. IMPORTANCE: Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli, thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.

Phage-mediated colistin resistance in Acinetobacter baumannii.

AIMS: Antimicrobial resistance is a global threat to human health, and Acinetobacter baumannii is a paradigmatic example of how rapidly bacteria become resistant to clinically relevant antimicrobials. The emergence of multidrug-resistant A. baumannii strains has forced the revival of colistin as a last-resort drug, suddenly leading to the emergence of colistin resistance. We investigated the genetic and molecular basis of colistin resistance in A. baumannii, and the mechanisms implicated in its regulation and dissemination. METHODS: Comparative genomic analysis was combined with genetic, biochemical, and phenotypic assays to characterize Φ19606, an A. baumannii temperate bacteriophage that carries a colistin resistance gene. RESULTS: Ф19606 was detected in 41% of 523 A. baumannii complete genomes and demonstrated to act as a mobile vehicle of the colistin resistance gene eptA1, encoding a functional lipid A phosphoethanolamine transferase. The eptA1 gene is coregulated with its chromosomal homolog pmrC via the PmrAB two-component system and confers colistin resistance when induced by low calcium and magnesium levels. Resistance selection assays showed that the eptA1-harbouring phage Ф19606 promotes the emergence of spontaneous colistin-resistant mutants. CONCLUSIONS: Φ19606 is an unprecedented example of a self-transmissible phage vector implicated in the dissemination of colistin resistance.

The antimicrobial peptide Esc(1-21)-1c increases susceptibility of Pseudomonas aeruginosa to conventional antibiotics by decreasing the expression of the MexAB-OprM efflux pump.

Introduction: The increase in bacterial strains resistant to conventional antibiotics is an alarming problem for human health and could lead to pandemics in the future. Among bacterial pathogens responsible for a large variety of severe infections there is Pseudomonas aeruginosa. Therefore, there is an urgent need for new molecules with antimicrobial activity or that can act as adjuvants of antibiotics already in use. In this scenario, antimicrobial peptides (AMPs) hold great promise. Recently, we characterized a frog-skin AMP derived from esculentin-1a, namely Esc(1-21)-1c, endowed with antipseudomonal activity without being cytotoxic to human cells. Methods: The combinatorial effect of the peptide and antibiotics was investigated through the checkerboard assay, differential proteomic and transcriptional analysis. Results: Here, we found that Esc(1-21)-1c can synergistically inhibit the growth of P. aeruginosa cells with three different antibiotics, including tetracycline. We therefore investigated the underlying mechanism implemented by the peptide using a differential proteomic approach. The data revealed a significant decrease in the production of three proteins belonging to the MexAB-OprM efflux pump upon treatment with sub-inhibitory concentration of Esc(1-21)-1c. Down-regulation of these proteins was confirmed by transcriptional analysis and direct measurement of their relative levels in bacterial cells by tandem mass spectrometry analysis in multiple reaction monitoring scan mode. Conclusion: These evidences suggest that treatment with Esc(1-21)-1c in combination with antibiotics would increase the intracellular drug content making bacteria more susceptible to the antibiotic. Overall, these results highlight the importance of characterizing new molecules able to synergize with conventional antibiotics, paving the way for the development of alternative therapeutic strategies based on AMP/antibiotic formulations to counteract the emergence of resistant bacterial strains and increase the use of "old" antibiotics in medical practice.

RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing.

In its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that considerable cell-to-cell variation in the QS activation state occurs during growth, often resulting in coexisting subpopulations of cells in which QS is active (quorate cells) or inactive (non-quorate cells). Heterogeneity has been observed in the las QS system of the opportunistic pathogen Pseudomonas aeruginosa. However, the molecular mechanisms underlying this phenomenon have not yet been defined. The las QS system consists of an incoherent feedforward loop in which the LasR transcriptional regulator activates the expression of the lasI synthase gene and rsaL, coding for the lasI transcriptional repressor RsaL. Here, single-cell-level gene expression analyses performed in ad hoc engineered biosensor strains and deletion mutants revealed that direct binding of RsaL to the lasI promoter region increases heterogeneous activation of the las QS system. Experiments performed with a dual-fluorescence reporter system showed that the LasR-dependent expression of lasI and rsaL does not correlate in single cells, indicating that RsaL acts as a brake that stochastically limits the transition of non-quorate cells to the quorate state in a subpopulation of cells expressing high levels of this negative regulator. Interestingly, the rhl QS system that is not controlled by an analogous RsaL protein showed higher homogeneity with respect to the las system. IMPORTANCE Single-cell analyses can reveal that despite experiencing identical physico-chemical conditions, individual bacterial cells within a monoclonal population may exhibit variations in gene expression. Such phenotypic heterogeneity has been described for several aspects of bacterial physiology, including QS activation. This study demonstrates that the transition of non-quorate cells to the quorate state is a graded process that does not occur at a specific cell density and that subpopulations of non-quorate cells also persist at high cell density. Here, we provide a mechanistic explanation for this phenomenon, showing that a negative feedback regulatory loop integrated into the las system has a pivotal role in promoting cell-to-cell variation in the QS activation state and in limiting the transition of non-quorate cells to the quorate state in P. aeruginosa.

Alkyl-quinolone-dependent quorum sensing controls prophage-mediated autolysis in Pseudomonas aeruginosa colony biofilms.

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.

Synergistic Activity of Colistin in Combination with Clofoctol against Colistin Resistant Gram-Negative Pathogens.

Colistin is a bactericidal antibiotic identified decades ago which is active against a number of Gram-negative pathogens. After early elimination from clinical use due to toxicity issues, colistin has been reintroduced as a last-resort treatment for antibiotic-resistant Gram-negative infections lacking other therapeutic options. Inevitably, colistin resistance has emerged among clinical isolates, making the development of colistin adjuvants extremely beneficial. Clofoctol is a synthetic antibiotic active against Gram-positive bacteria, with low toxicity and high tropism for the airways. Interestingly, clofoctol has been found to have multiple biological activities and has been proposed for the treatment of several obstructive lung diseases, including asthma, lung cancer, and SARS-CoV-2 infection. In this study, the activity of clofoctol as a colistin adjuvant was investigated in Gram-negative lung pathogens that are critical for the high prevalence of multidrug-resistant isolates, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Clofoctol potentiated the bactericidal effect of colistin in all tested strains and reduced colistin MICs below the susceptibility breakpoint in nearly all colistin-resistant strains. Overall, this observation supports the development of inhaled clofoctol-colistin formulations for the treatment of difficult-to-treat airway infections caused by Gram-negative pathogens. IMPORTANCE Colistin is used as a last-resort antibiotic against extensively drug-resistant Gram-negative pathogens. However, colistin resistance is on the rise. Clofoctol is an antibiotic used against Gram-positive bacteria, with low toxicity and high penetration and storage in the airways. Here, a strong synergistic activity of the colistin-clofoctol combination against colistin-resistant Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii isolates is reported, supporting the development of clofoctol-colistin formulations for the therapy of difficult-to-treat airways infections caused by these Gram-negative pathogens.

The Pseudomonas aeruginosa DksA1 protein is involved in H2O2 tolerance and within-macrophages survival and can be replaced by DksA2.

In Gram-negative pathogens, the stringent response regulator DksA controls the expression of hundreds of genes, including virulence-related genes. Interestingly, Pseudomonas aeruginosa has two functional DksA paralogs: DksA1 is constitutively expressed and has a zinc-finger motif, while DksA2 is expressed only under zinc starvation conditions and does not contain zinc. DksA1 stimulates the production of virulence factors in vitro and is required for full pathogenicity in vivo. DksA2 can replace these DksA1 functions. Here, the role of dksA paralogs in P. aeruginosa tolerance to H2O2-induced oxidative stress has been investigated. The P. aeruginosa dksA1 dksA2 mutant showed impaired H2O2 tolerance in planktonic and biofilm-growing cultures and increased susceptibility to macrophages-mediated killing compared to the wild type. Complementation with either dksA1 or dksA2 genes restored the wild type phenotypes. The DksA-dependent tolerance to oxidative stress involves, at least in part, the positive transcriptional control of both katA and katE catalase-encoding genes. These data support the hypothesis that DksA1 and DksA2 are eco-paralogs with indistinguishable function but optimal activity under different environmental conditions, and highlight their mutual contribution to P. aeruginosa virulence.

In vitro Activity of Antivirulence Drugs Targeting the las or pqs Quorum Sensing Against Cystic Fibrosis Pseudomonas aeruginosa Isolates.

The chronic lung infection caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. Antivirulence drugs targeting P. aeruginosa quorum sensing (QS) systems are intensively studied as antibiotics substitutes or adjuvants. Previous studies, carried out in non-CF P. aeruginosa reference strains, showed that the old drugs niclosamide and clofoctol could be successfully repurposed as antivirulence drugs targeting the las and pqs QS systems, respectively. However, frequent emergence of QS-defective mutants in the CF lung undermines the use of QS inhibitors in CF therapy. Here, QS signal production and susceptibility to niclosamide and clofoctol have been investigated in 100 P. aeruginosa CF isolates, with the aim of broadening current knowledge on the potential of anti-QS compounds in CF therapy. Results showed that 85, 78, and 69% of the CF isolates from our collection were proficient for the pqs, rhl, and las QS systems, respectively. The ability of both niclosamide and clofoctol to inhibit QS and virulence in vitro was highly variable and strain-dependent. Niclosamide showed an overall low range of activity and its negative effect on las signal production did not correlate with a decreased production of virulence factors. On the other hand, clofoctol displayed a broader QS inhibitory effect in CF isolates, with consequent reduction of the pqs-controlled virulence factor pyocyanin. Overall, this study highlights the importance of testing new antivirulence drugs against large panels of P. aeruginosa CF clinical isolates before proceeding to further pre-clinical studies and corroborates previous evidence that strains naturally resistant to QS inhibitors occur among CF isolates. However, it is also shown that resistance to pqs inhibitors is less frequent than resistance to las inhibitors, thus supporting the development of pqs inhibitors for antivirulence therapy in CF.

PqsE Expands and Differentially Modulates the RhlR Quorum Sensing Regulon in Pseudomonas aeruginosa.

In the opportunistic pathogen Pseudomonas aeruginosa, many virulence traits are finely regulated by quorum sensing (QS), an intercellular communication system that allows the cells of a population to coordinate gene expression in response to cell density. The key aspects underlying the functionality of the complex regulatory network governing QS in P. aeruginosa are still poorly understood, including the interplay between the effector protein PqsE and the transcriptional regulator RhlR in controlling the QS regulon. Different studies have focused on the characterization of PqsE- and RhlR-controlled genes in genetic backgrounds in which RhlR activity can be modulated by PqsE and pqsE expression is controlled by RhlR, thus hampering identification of the distinct regulons controlled by PqsE and RhlR. In this study, a P. aeruginosa PAO1 mutant strain with deletion of multiple QS elements and inducible expression of pqsE and/or rhlR was generated and validated. Transcriptomic analyses performed on this genetic background allowed us to unambiguously define the regulons controlled by PqsE and RhlR when produced alone or in combination. Transcriptomic data were validated via reverse transcription-quantitative PCR (RT-qPCR) and transcriptional fusions. Overall, our results showed that PqsE has a negligible effect on the P. aeruginosa transcriptome in the absence of RhlR, and that multiple RhlR subregulons exist with distinct dependency on PqsE. Overall, this study contributes to untangling the regulatory link between the pqs and rhl QS systems mediated by PqsE and RhlR and clarifying the impact of these QS elements on the P. aeruginosa transcriptome. IMPORTANCE 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. While it is known that PqsE can stimulate the ability of RhlR to control some virulence factors, no data are available to allow clear discrimination of the PqsE and RhlR regulons. The data produced in this study demonstrate that PqsE mainly impacts the P. aeruginosa transcriptome via an RhlR-dependent pathway and splits the RhlR regulon into PqsE-dependent and PqsE-independent subregulons. Besides contributing to untangling of the complex QS network of P. aeruginosa, our data confirm that both PqsE and RhlR are suitable targets for the development of antivirulence drugs.

Effect of a Defective Clamp Loader Complex of DNA Polymerase III on Growth and SOS Response in Pseudomonas aeruginosa.

DNA polymerase III (Pol III) is the replicative enzyme in bacteria. It consists of three subcomplexes, the catalytic core, the ß clamp, and the clamp loader. While this complex has been thoroughly characterized in the model organism Escherichia coli, much less is known about its functioning and/or its specific properties in other bacteria. Biochemical studies highlighted specific features in the clamp loader subunit ψ of Pseudomonas aeruginosa as compared to its E. coli counterpart, and transposon mutagenesis projects identified the ψ-encoding gene holD among the strictly essential core genes of P. aeruginosa. By generating a P. aeruginosa holD conditional mutant, here we demonstrate that, as previously observed for E. coli holD mutants, HolD-depleted P. aeruginosa cells show strongly decreased growth, induction of the SOS response, and emergence of suppressor mutants at high frequency. However, differently from what was observed in E. coli, the growth of P. aeruginosa cells lacking HolD cannot be rescued by the deletion of genes for specialized DNA polymerases. We also observed that the residual growth of HolD-depleted cells is strictly dependent on homologous recombination functions, suggesting that recombination-mediated rescue of stalled replication forks is crucial to support replication by a ψ-deficient Pol III enzyme in P. aeruginosa.

Variable Susceptibility to Gallium Compounds of Major Cystic Fibrosis Pathogens.

The decreasing efficacy of existing antibiotics against pulmonary pathogens that affect cystic fibrosis (CF) patients calls for the development of novel antimicrobials. Iron uptake and metabolism are vital processes for bacteria, hence potential therapeutic targets. Gallium [Ga(III)] is a ferric iron-mimetic that inhibits bacterial growth by disrupting iron uptake and metabolism. In this work we evaluate the efficacy of three Ga(III) compounds, namely, Ga(NO3)3, (GaN), Ga(III)-maltolate (GaM), and Ga(III)-protoporphyrin IX (GaPPIX), against a collection of CF pathogens using both reference media and media mimicking biological fluids. All CF pathogens, except Streptococcus pneumoniae, were susceptible to at least one Ga(III) compound. Notably, Mycobacterium abscessus and Stenotrophomonas maltophilia were susceptible to all Ga(III) compounds. Achromobacter xylosoxidans, Burkholderia cepacia complex, and Pseudomonas aeruginosa were more susceptible to GaN and GaM, whereas Staphylococcus aureus and Haemophilus influenzae were more sensitive to GaPPIX. The results of this study support the development of Ga(III)-based therapy as a broad-spectrum strategy to treat CF lung infections.

The two Pseudomonas aeruginosa DksA stringent response proteins are largely interchangeable at the whole transcriptome level and in the control of virulence-related traits.

The stringent response regulator DksA plays a key role in Gram negative bacteria adaptation to challenging environments. Intriguingly, the plant and human pathogen Pseudomonas aeruginosa is unique as it expresses two functional DksA paralogs: DksA1 and DksA2. However, the role of DksA2 in P. aeruginosa adaptive strategies has been poorly investigated so far. Here, RNA-Seq analysis and phenotypic assays showed that P. aeruginosa DksA1 and DksA2 proteins are largely interchangeable. Relative to wild type P. aeruginosa, transcription of 1779 genes was altered in a dksA1 dksA2 double mutant, and the wild type expression level of ≥90% of these genes was restored by in trans complementation with either dksA1 or dksA2. Interestingly, the expression of a small sub-set of genes seems to be preferentially or exclusively complemented by either dksA1 or dksA2. In addition, evidence has been provided that the DksA-dependent regulation of virulence genes expression is independent and hierarchically dominant over two major P. aeruginosa regulatory circuits, i.e., quorum sensing and cyclic-di-GMP signalling systems. Our findings support the prominent role of both DksA paralogs in P. aeruginosa environmental adaptation.

Generation of Genetic Tools for Gauging Multiple-Gene Expression at the Single-Cell Level.

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple-gene expression at the single-cell level has limited the understanding of phenotypic heterogeneity. To investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa, a genetic tool that allows gauging multiple-gene expression at the single-cell level has been generated. This tool, named pRGC, consists of a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP), and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single-cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population without the need for image processing for spectral cross talk correction. In addition, two pRGC variants have been generated for either (i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome that is suitable for long-term experiments in the absence of antibiotic selection or (ii) replication in bacterial genera other than Pseudomonas The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple-gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity.IMPORTANCE Within a bacterial population, single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial subpopulations with distinct phenotypes. The analysis of gene expression at the single-cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation, and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple-gene expression at the single-cell level by fluorescence microscopy without the need for advanced image-processing procedures. A proof of concept has been provided by investigating iron uptake and iron storage gene expression in response to iron availability in P. aeruginosa.

Identification of FDA-approved antivirulence drugs targeting the Pseudomonas aeruginosa quorum sensing effector protein PqsE.

The ability of the bacterial pathogen Pseudomonas aeruginosa to cause both chronic and acute infections mainly relies on its capacity to finely modulate the expression of virulence factors through a complex network of regulatory circuits, including the pqs quorum sensing (QS) system. While in most QS systems the signal molecule/receptor complexes act as global regulators that modulate the expression of QS-controlled genes, the main effector protein of the pqs system is PqsE. This protein is involved in the synthesis of the QS signal molecules 2-alkyl-4(1H)-quinolones (AQs), but it also modulates the expression of genes involved in virulence factors production and biofilm formation via AQ-independent pathway(s). P. aeruginosa pqsE mutants disclose attenuated virulence in plant and animal infection models, hence PqsE is considered a good target for the development of antivirulence drugs against P. aeruginosa. In this study, the negative regulation exerted by PqsE on its own transcription has been exploited to develop a screening system for the identification of PqsE inhibitors in a library of FDA-approved drugs. This led to the identification of nitrofurazone and erythromycin estolate, two antibiotic compounds that reduce the expression of PqsE-dependent virulence traits and biofilm formation in the model strain P. aeruginosa PAO1 at concentrations far below those affecting the bacterial growth rate. Notably, both drugs reduce the production of the PqsE-controlled virulence factor pyocyanin also in P. aeruginosa strains isolated from cystic fibrosis patients, and do not antagonize the activity of antibiotics commonly used to treat P. aeruginosa infection.

Adaptive tracking of enzymatic reactions with quantum light.

Enzymes are essential to maintain organisms alive. Some of the reactions they catalyze are associated with a change in reagents chirality, hence their activity can be tracked by using optical means. However, illumination affects enzyme activity: the challenge is to operate at low-intensity regime avoiding loss in sensitivity. Here we apply quantum phase estimation to real-time measurement of invertase enzymatic activity. Control of the probe at the quantum level demonstrates the potential for reducing invasiveness with optimized sensitivity at once. This preliminary effort, bringing together methods of quantum physics and biology, constitutes an important step towards full development of quantum sensors for biological systems.

In silico Selection and Experimental Validation of FDA-Approved Drugs as Anti-quorum Sensing Agents.

The emergence of antibiotic resistant bacterial pathogens is increasing at an unprecedented pace, calling for the development of new therapeutic options. Small molecules interfering with virulence processes rather than growth hold promise as an alternative to conventional antibiotics. Anti-virulence agents are expected to decrease bacterial virulence and to pose reduced selective pressure for the emergence of resistance. In the opportunistic pathogen Pseudomonas aeruginosa the expression of key virulence traits is controlled by quorum sensing (QS), an intercellular communication process that coordinates gene expression at the population level. Hence, QS inhibitors represent promising anti-virulence agents against P. aeruginosa. Virtual screenings allow fast and cost-effective selection of target ligands among vast libraries of molecules, thus accelerating the time and limiting the cost of conventional drug-discovery processes, while the drug-repurposing approach is based on the identification of off-target activity of FDA-approved drugs, likely endowed with low cytotoxicity and favorable pharmacological properties. This study aims at combining the advantages of virtual screening and drug-repurposing approaches to identify new QS inhibitors targeting the pqs QS system of P. aeruginosa. An in silico library of 1,467 FDA-approved drugs has been screened by molecular docking, and 5 hits showing the highest predicted binding affinity for the pqs QS receptor PqsR (also known as MvfR) have been selected. In vitro experiments have been performed by engineering ad hoc biosensor strains, which were used to verify the ability of hit compounds to decrease PqsR activity in P. aeruginosa. Phenotypic analyses confirmed the impact of the most promising hit, the antipsychotic drug pimozide, on the expression of P. aeruginosa PqsR-controlled virulence traits. Overall, this study highlights the potential of virtual screening campaigns of FDA-approved drugs to rapidly select new inhibitors of important bacterial functions.

New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors.

The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model.IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.

Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa.

The rise in antibiotic resistance among bacterial pathogens has prompted the exploitation of alternative antibacterial strategies, such as antivirulence therapy. By inhibiting virulence traits, antivirulence drugs are expected to lessen pathogenicity without affecting bacterial growth, therefore avoiding the spread of resistance. However, some studies argued against this assumption, and the lack of antivirulence drugs in clinical use hampers the empirical assessment of this concept. Here we compared the mode of action and range of activity of two drugs which have been proposed for repurposing as quorum sensing and pyoverdine inhibitors in the human pathogen Pseudomonas aeruginosa: the anticancer drug 5-fluorouracil (5-FU) and the antimycotic drug 5-fluorocytosine (5-FC), respectively. The effect on bacterial growth, emergence and spread of resistance, and activity against clinical isolates were assessed. Our results confirm that 5-FU has growth inhibitory activity on reference strains and can rapidly select for spontaneous resistant mutants with loss-of-function mutations in the upp gene, responsible for uracil conversion into UMP. These mutants were also insensitive to the anti-pyoverdine effect of 5-FC. Conversely, 5-FC did not cause relevant growth inhibition, likely because of poor enzymatic conversion into 5-FU by cytosine deaminase. However, coculturing experiments showed that 5-FU resistant mutants can outcompete sensitive cells in mixed populations, in the presence of not only 5-FU but also 5-FC. Moreover, we observed that serial passages of wild-type cells in 5-FC-containing medium leads to the appearance and spread of 5-FC insensitive sub-populations of 5-FU resistant cells. The different effect on growth of 5-FU and 5-FC was overall conserved in a large collection of cystic fibrosis (CF) isolates, corresponding to different infection stages and antibiotic resistance profiles, although high variability was observed among strains. Notably, this analysis also revealed a significant number of pyoverdine-deficient isolates, whose proportion apparently increases over the course of the CF infection. This study demonstrates that the efficacy of an antivirulence drug with no apparent effect on growth can be significantly influenced by the emergence of insensitive mutants, and highlights the importance of the assessment of resistance-associated fitness cost and activity on clinical isolates for the development of "resistance-proof" antivirulence drugs.