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 Fe 3؉ 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 Fe 2؉ 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 Fe 3؉ 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.

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.

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.

b 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(NO 3 ) 3 in inhibiting P. aeruginosa growth, and its activity was dependent on increased Ga entrance into the cell through the pyochelin translocon.I ron (Fe) is an essential nutrient for nearly all forms of life, being the cofactor of many vital enzymes involved in DNA synthesis, metabolism, and the oxidative stress response (1). Pathogenic bacteria must counteract an Fe-poor environment during infection, since Fe is unavailable to invading pathogens due to sequestration by the Fe carrier and storage proteins of the host (2). Bacteria have evolved multiple strategies to acquire Fe from the host, the most common being through the production of siderophores. These compounds are secreted in the extracellular milieu, where they form stable complexes with Fe and other transition metals (depending on their coordination chemistry) and convey the metal to the bacterial cell via specific active transport systems (3). Given the importance of Fe in bacterial metabolism and the paucity of effective antibiotics for multidrug-resistant bacteria, Fe uptake and metabolism have recently been assessed as targets for the development of new antibacterials (4-7).Gallium (Ga III ) is a semimetal that shares a number of chemical similarities with the oxidized Fe form (Fe III 3 , the active component of the FDA-approved formulation Ganite (Genta), have been investigated in a number of species (recently reviewed in references 9-12). In particular, Ga III inhibits both planktonic and biofilm growth of the opportunistic pathogen Pseudomonas aeruginosa and causes significant protection from P. aeruginosa infection in animal models (13,14).In the present study, we attempted to improve the antibacterial activity of Ga III on P. aeruginosa by complexation with suitable carriers (either synthetic chelators or siderophores) that are actively taken up by the bacterium and that stimulate, to a variable extent, its growth under conditions of extreme Fe deficiency (see Fig. S1 in the supplemental material). Both the P. aeruginosa reference strain PAO1 and the cystic fibrosis isolate TR1 (15) were used for growth promotion/inhibition assays. Ga III -chelator complexes were generated by mixing, in the appropriate ratios (Fig. 1), aqueous solutions of Ga(NO 3 ) 3 with ferrichrome (FER) (Sigma), sodium dicitrate (CIT) (Sigma), desferrioxamine (DFO) (Novartis), sodium salicylate (SAL) (Sigma), and the autologous siderophores pyoverdine (PVD) and pyochelin (PCH). PVD and PCH were purified from culture supernatants of a PCH-defective P. aeruginosa mutant (PAO1⌬pchD; see Table S1 and Supplemental Experimental Procedures in the supplemental material) and a PVD-defective P. aeruginosa mutant (PAO1⌬pvdA) ...

In Pseudomonas aeruginosa the Gac signaling system and the second messenger cyclic diguanylate (c-di-GMP) participate in the control of the switch between planktonic and biofilm lifestyles, by regulating the production of the two exopolysaccharides Pel and Psl. The Gac and c-di-GMP regulatory networks also coordinately promote the production of the pyoverdine siderophore, and the extracellular polysaccharides Pel and Psl have recently been found to mediate c-di-GMP-dependent regulation of pyoverdine genes. Here we demonstrate that Pel and Psl are also essential for Gac–mediated activation of pyoverdine production. A pel psl double mutant produces very low levels of pyoverdine and shows a marked reduction in the expression of the pyoverdine-dependent virulence factors exotoxin A and PrpL protease. While the exopolysaccharide-proficient parent strain forms multicellular planktonic aggregates in liquid cultures, the Pel and Psl-deficient mutant mainly grows as dispersed cells. Notably, artificially induced cell aggregation is able to restore pyoverdine-dependent gene expression in the pel psl mutant, in a way that appears to be independent of iron diffusion or siderophore signaling, as well as of recently described contact-dependent mechanosensitive systems. This study demonstrates that cell aggregation represents an important cue triggering the expression of pyoverdine-related genes in P. aeruginosa, suggesting a novel link between virulence gene expression, cell–cell interaction and the multicellular community lifestyle.