Emanuela Frangipani

Repurposing the antimycotic drug flucytosine for suppression of Pseudomonas aeruginosa pathogenicity

Although antibiotic resistance represents a public health emergency, the pipeline of new antibiotics is running dry. Repurposing of old drugs for new clinical applications is an attractive strategy for drug development. We used the bacterial pathogen Pseudomonas aeruginosa as a target for the screening of antivirulence activity among marketed drugs. We found that the antimycotic agent flucytosine inhibits the expression of the iron-starvation σ-factor PvdS, thereby repressing the production of major P. aeruginosa virulence factors, namely pyoverdine, PrpL protease, and exotoxin A. Flucytosine administration at clinically meaningful dosing regimens suppressed P. aeruginosa pathogenicity in a mouse model of lung infection. The in vitro and in vivo activity of flucytosine against P. aeruginosa, combined with its desirable pharmacological properties, paves the way for clinical trials on the anti-P. aeruginosa efficacy of flucytosine in humans.

The Gac/Rsm and cyclic-di-GMP signalling networks coordinately regulate iron uptake in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a versatile bacterial pathogen capable of occupying diverse ecological niches. To cope with iron limitation, P. aeruginosa secretes two siderophores, pyoverdine and pyochelin, whose ability to deliver iron to the cell is crucial for biofilm formation and pathogenicity. In this study, we describe a link between iron uptake and the Gac/Rsm system, a conserved signal transducing pathway of P. aeruginosa that controls the production of extracellular products and virulence factors, as well as the switch from planktonic to biofilm lifestyle. We have observed that pyoverdine and pyochelin production in P. aeruginosa is strongly dependent on the activation state of the Gac/Rsm pathway, which controls siderophore regulatory and biosynthetic genes at the transcriptional level, in a manner that does not involve regulation of ferric uptake regulator (Fur) expression. Gac/Rsm-mediated regulation of iron uptake genes appears to be conserved in different P. aeruginosa strains. Further experiments led to propose that the Gac/Rsm system regulates siderophore production through modulation of the intracellular levels of the second messenger c-di-GMP, indicating that the c-di-GMP and the Gac/Rsm regulatory networks essential for biofilm formation can also coordinately control iron uptake in P. aeruginosa.

Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

ABSTRACT Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe3+ uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe2+ acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+ transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities.

Repurposing of gallium-based drugs for antibacterial therapy

While the occurrence and spread of antibiotic resistance in bacterial pathogens is vanishing current anti‐infective therapies, the antibiotic discovery pipeline is drying up. In the last years, the repurposing of existing drugs for new clinical applications has become a major research area in drug discovery, also in the field of anti‐infectives. This review discusses the potential of repurposing previously approved gallium formulations in antibacterial chemotherapy. Gallium has no proven function in biological systems, but it can act as an iron‐mimetic in both prokaryotic and eukaryotic cells. The activity of gallium mostly relies on its ability to replace iron in redox enzymes, thus impairing their function and ultimately hampering cell growth. Cancer cells and bacteria are preferential gallium targets due to their active metabolism and fast growth. The wealth of knowledge on the pharmacological properties of gallium has opened the door to the repurposing of gallium‐based drugs for the treatment of infections sustained by antibiotic‐resistant bacterial pathogens, such as Acinetobacter baumannii or Pseudomonas aeruginosa, and for suppression of Mycobacterium tuberculosis growth. The promising antibacterial activity of gallium both in vitro and in different animal models of infection raises the hope that gallium will confirm its efficacy in clinical trials, and will become a valuable therapeutic option to cure otherwise untreatable bacterial infections.

Promises and failures of gallium as an antibacterial agent.

Gallium has a long history as a diagnostic and chemotherapeutic agent. The pharmacological properties of Ga(III) rely on chemical mimicry; when Ga(III) is exogenously supplied to living cells it can replace Fe(III) within target molecules, thereby perturbing bacterial metabolism. Ga(III)-induced metabolic distresses are dramatic in fast-growing cells, like bacterial cells. Interest in the antibacterial properties of Ga(III) has been raised by the compelling need for novel drugs to combat multidrug-resistant bacteria and by the shortage of new antibiotic candidates in the pharmaceutical pipeline. Ga(III) activity has been demonstrated, both in vitro and in animal models of infections, on several bacterial pathogens, also including intracellular and biofilm-forming bacteria. Ga(III) activity is affected by iron availability and the metabolic state of the cell, being maximal in iron-poor media and in respiring cells. Synergism between Ga(III) and antibiotics holds promise as last resort therapy for infections sustained by pandrug-resistant bacteria.

Involvement of Pseudomonas aeruginosa Rhodanese in Protection from Cyanide Toxicity

ABSTRACT Cyanide is a serious environmental pollutant and a biocontrol metabolite in plant growth-promoting Pseudomonas species. Here we report on the presence of multiple sulfurtransferases in the cyanogenic bacterium Pseudomonas aeruginosa PAO1 and investigate in detail RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) which converts cyanide to less toxic thiocyanate. RhdA is a cytoplasmic enzyme acting as the principal rhodanese in P. aeruginosa. The rhdA gene forms a transcriptional unit with the PA4955 and psd genes and is controlled by two promoters located upstream of PA4955 and rhdA. Both promoters direct constitutive RhdA expression and show similar patterns of activity, involving moderate down-regulation at the stationary phase or in the presence of exogenous cyanide. We previously observed that RhdA overproduction protects Escherichia coli against cyanide toxicity, and here we show that physiological RhdA levels contribute to P. aeruginosa survival under cyanogenic conditions. The growth of a ΔrhdA mutant is impaired under cyanogenic conditions and fully restored upon complementation with rhdA. Wild-type P. aeruginosa outcompetes the ΔrhdA mutant in cyanogenic coculture assays. Hence, RhdA could be regarded as an effector of P. aeruginosa intrinsic resistance to cyanide, insofar as it provides the bacterium with a defense mechanism against endogenous cyanide toxicity, in addition to cyanide-resistant respiration.

Copper acquisition by the SenC protein regulates aerobic respiration in Pseudomonas aeruginosa PAO1

Aerobic respiration of Pseudomonas aeruginosa involves four terminal oxidases belonging to the heme-copper family (that is, three cytochrome c oxidases and one quinol oxidase) plus one copper-independent, cyanide-insensitive quinol oxidase (CIO). The PA0114 gene encoding an SCO1/SenC-type protein, which is known to be important for copper delivery to cytochrome c in yeast, Rhodobacter spp. and Agrobacterium tumefaciens, was found to be important for copper acquisition and aerobic respiration in P. aeruginosa. A PA0114 (senC) mutant grew poorly in low-copper media and had low cytochrome cbb(3)-type oxidase activity, but expressed CIO at increased levels, by comparison with the wild-type PAO1. Addition of copper reversed these phenotypes, suggesting that periplasmic copper capture by the SenC protein helps P. aeruginosa to adapt to copper deprivation.

Unravelling the Genome-Wide Contributions of Specific 2-Alkyl-4-Quinolones and PqsE to Quorum Sensing in Pseudomonas aeruginosa

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.

A putative de- N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance in Pseudomonas aeruginosa

A major cause of treatment failure of infections caused by Pseudomonas aeruginosa is the presence of antibiotic-insensitive persister cells. The mechanism of persister formation in P. aeruginosa is largely unknown, and so far, only few genetic determinants have been linked to P. aeruginosa persistence. Based on a previous high-throughput screening, we here present dnpA (de-N-acetylase involved in persistence; gene locus PA14_66140/PA5002) as a new gene involved in noninherited fluoroquinolone tolerance in P. aeruginosa. Fluoroquinolone tolerance of a dnpA mutant is strongly reduced both in planktonic culture and in a biofilm model, whereas overexpression of dnpA in the wild-type strain increases the persister fraction. In addition, the susceptibility of the dnpA mutant to different classes of antibiotics is not affected. dnpA is part of the conserved LPS core oligosaccharide biosynthesis gene cluster. Based on primary sequence analysis, we predict that DnpA is a de-N-acetylase, acting on an unidentified substrate. Site-directed mutagenesis suggests that this enzymatic activity is essential for DnpA-mediated persistence. A transcriptome analysis indicates that DnpA primarily affects the expression of genes involved in surface-associated processes. We discuss the implications of these findings for future antipersister therapies targeted at chronic P. aeruginosa infections.

Pyochelin potentiates the inhibitory activity of gallium on Pseudomonas aeruginosa

ABSTRACT Gallium (Ga) is an iron mimetic that has successfully been repurposed for antibacterial chemotherapy. To improve the antibacterial potency of Ga on Pseudomonas aeruginosa, the effect of complexation with a variety of siderophores and synthetic chelators was tested. Ga complexed with the pyochelin siderophore (at a 1:2 ratio) was more efficient than Ga(NO3)3 in inhibiting P. aeruginosa growth, and its activity was dependent on increased Ga entrance into the cell through the pyochelin translocon.