Intermicrobial interaction: Aspergillus fumigatus siderophores protect against competition by Pseudomonas aeruginosa.

Pseudomonas aeruginosa and Aspergillus fumigatus are pathogens frequently co-inhabiting immunocompromised patient airways, particularly in people with cystic fibrosis. Both microbes depend on the availability of iron, and compete for iron in their microenvironment. We showed previously that the P. aeruginosa siderophore pyoverdine is the main instrument in battling A. fumigatus biofilms, by iron chelation and denial of iron to the fungus. Here we show that A. fumigatus siderophores defend against anti-fungal P. aeruginosa effects. P. aeruginosa supernatants produced in the presence of wildtype A. fumigatus planktonic supernatants (Afsup) showed less activity against A. fumigatus biofilms than P. aeruginosa supernatants without Afsup, despite higher production of pyoverdine by P. aeruginosa. Supernatants of A. fumigatus cultures lacking the sidA gene (AfΔsidA), unable to produce hydroxamate siderophores, were less capable of protecting A. fumigatus biofilms from P. aeruginosa supernatants and pyoverdine. AfΔsidA biofilm was more sensitive towards inhibitory effects of pyoverdine, the iron chelator deferiprone (DFP), or amphothericin B than wildtype A. fumigatus biofilm. Supplementation of sidA-deficient A. fumigatus biofilm with A. fumigatus siderophores restored resistance to pyoverdine. The A. fumigatus siderophore production inhibitor celastrol sensitized wildtype A. fumigatus biofilms towards the anti-fungal activity of DFP. In conclusion, A. fumigatus hydroxamate siderophores play a pivotal role in A. fumigatus competition for iron against P. aeruginosa.

[Iron and invasive fungal infection]. / Hierro e infección fúngica invasiva.

Iron is an essential factor for both the growth and virulence of most of microorganisms. As a part of the innate (or nutritional) immune system, mammals have developed different mechanisms to store and transport this element in order to limit free iron bioavailability. To survive in this hostile environment, pathogenic fungi have specific uptake systems for host iron sources, one of the most important of which is based on the synthesis of siderophores-soluble, low-molecular-mass, high-affinity iron chelators. The increase in free iron that results from iron-overload conditions is a well-established risk factor for invasive fungal infection (IFI) such as mucormycosis or aspergillosis. Therefore, iron chelation may be an appealing therapeutic option for these infections. Nevertheless, deferoxamine -the first approved iron chelator- paradoxically increases the incidence of IFI, as it serves as a xeno-siderophore to Mucorales. On the contrary, the new oral iron chelators (deferiprone and deferasirox) have shown to exert a deleterious effect on fungal growth both in vitro and in animal models. The present review focuses on the role of iron metabolism in the pathogenesis of IFI and summarises the preclinical data, as well as the limited clinical experience so far, in the use of new iron chelators as treatment for mucormycosis and invasive aspergillosis.

Catecholate siderophores protect bacteria from pyochelin toxicity.

BACKGROUND: Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition. METHODS AND PRINCIPAL FINDINGS: Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity. CONCLUSIONS: We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.

The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth.

Many species of mycobacteria form structured biofilm communities at liquid-air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 microM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron-responsive genes - especially those involved in siderophore synthesis and iron uptake - are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 microM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron-exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 microM) conditions. A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.

Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin M114 siderophore.

In the soil bacterium Pseudomonas fluorescens M114, extracellular proteolytic activity and fluorescent siderophore (pseudobactin M114) production were previously shown to be co-ordinately negatively regulated in response to environmental iron levels. An iron-starvation extracytoplasmic function sigma factor, PbrA, required for the transcription of siderophore biosynthetic genes, was also implicated in M114 protease regulation. The current study centred on the characterization and genetic regulation of the gene(s) responsible for protease production in M114. A serralysin-type metalloprotease gene, aprA, was identified and found to encode the major, if not only, extracellular protease produced by this strain. The expression of aprA and its protein product were found to be subject to complex regulation. Transcription analysis confirmed that PbrA was required for full aprA transcription under low iron conditions, while the ferric uptake regulator, Fur, was implicated in aprA repression under high iron conditions. Interestingly, the iron regulation of AprA was dependent on culture conditions, with PbrA-independent AprA-mediated proteolytic activity observed on skim milk agar supplemented with yeast extract, when supplied with iron or purified pseudobactin M114. These effects were not observed on skim milk agar without yeast extract. PbrA-independent aprA expression was also observed from a truncated transcriptional fusion when grown in sucrose asparagine tryptone broth supplied with iron or purified pseudobactin M114. Thus, experimental evidence suggested that iron mediated its effects via transcriptional activation by PbrA under low iron conditions, while an as-yet-unidentified sigma factor(s) may be required for the PbrA-independent aprA expression and AprA proteolytic activity induced by siderophore and iron.

Staphylococcus aureus siderophore-mediated iron-acquisition system plays a dominant and essential role in the utilization of transferrin-bound iron.

Staphylococcus aureus is known to be capable of utilizing transferrin-bound iron, via both siderophore- and transferrin-binding protein (named IsdA)-mediated iron-acquisition systems. This study was designed in order to determine which iron-acquisition system plays the essential or dominant role with respect to the acquisition of iron from human transferrin, in the growth of S. aureus. Holotransferrin (HT) and partially iron-saturated transferrin (PT), but not apotransferrin (AT), were found to stimulate the growth of S. aureus. S. aureus consumed most of the transferrin-bound iron during the exponential growth phase. Extracellular proteases were not, however, involved in the liberation of iron from transferrin. Transferrin-binding to the washed whole cells via IsdA was not observed during the culture. The expression of IsdA was observed only in the deferrated media with AT, but not in the media supplemented with PT or HT. In contrast, siderophores were definitely produced in the deferrated media with PT and HT, as well as in the media supplemented with AT. The siderophores proved to have the ability to remove iron directly from transferrin, but the washed whole cells expressing IsdA did not. In the bioassay, the growth of S. aureus on transferrin-bound iron was stimulated by the siderophores alone. These results demonstrate that the siderophore-mediated iron-acquisition system plays a dominant and essential role in the uptake of iron from transferrin, whereas the IsdA-mediated iron-acquisition system may play only an ancillary role in the uptake of iron from transferrin.

Pyoverdin is essential for virulence of Pseudomonas aeruginosa.

The role of pyoverdin, the main siderophore in iron-gathering capacity produced by Pseudomonas aeruginosa, in bacterial growth in vivo is controversial, although iron is important for virulence. To determine the ability of pyoverdin to compete for iron with the human iron-binding protein transferrin, wild-type P. aeruginosa ATCC 15692 (PAO1 strain) and PAO pyoverdin-deficient mutants were grown at 37 degrees C in bicarbonate-containing succinate medium to which apotransferrin had been added. Growth of the pyoverdin-deficient mutants was fully inhibited compared with that of the wild type but was restored when pyoverdin was added to the medium. Moreover, when growth took place at a temperature at which no pyoverdin production occurred (43 degrees C), the wild-type PAO1 strain behaved the same as the pyoverdin-deficient mutants, with growth inhibited by apotransferrin in the presence of bicarbonate and restored by pyoverdin supplementation. Growth inhibition was never observed in bicarbonate-free succinate medium, whatever the strain and the temperature for growth. In vivo, in contrast to results obtained with the wild-type strain, pyoverdin-deficient mutants demonstrated no virulence when injected at 10(2) CFU into burned mice. However, virulence was restored when purified pyoverdin originating from the wild-type strain was supplemented during the infection. These results strongly suggest that pyoverdin competes directly with transferrin for iron and that it is an essential element for in vivo iron gathering and virulence expression in P. aeruginosa. Rapid removal of iron from [59Fe]ferritransferrin by pyoverdin in vitro supports this view.

Acquisition of iron from transferrin and lactoferrin by the protozoan Leishmania chagasi.

Leishmania chagasi, the cause of South American visceral leishmaniasis, requires iron for its growth. However, the extent to which different iron sources can be utilized by the parasite is not known. To address this question, we studied acquisition of iron from lactoferrin and transferrin by the extracellular promastigote form of L. chagasi during growth in vitro. A promastigote growth medium based on minimal essential medium supplemented with iron-depleted serum supported promastigote growth only after the addition of exogenous iron. The addition of 8 microM iron chelated to lactoferrin or hemin resulted in normal promastigote growth. Ferritransferrin also supported promastigote growth, but only after a considerable lag. Promastigotes grown in all three iron sources generated similar amounts of hydroxyl radical upon exposure to hydrogen peroxide, indicating that none of these protected parasites against generation of this toxic radical. Promastigotes were able to take up 59Fe chelated to either transferrin or lactoferrin, although uptake from 59Fe-lactoferrin occurred more rapidly. 59Fe uptake from either 59Fe-transferrin or 59Fe-lactoferrin was inhibited by a 10-fold excess of unlabeled ferrilactoferrin, ferritransferrin, apolactoferrin, apotransferrin, or iron nitrilotriacetate but not ferritin or bovine serum albumin. There was no evidence for a role for parasite-derived siderophores or proteolytic cleavage of ferritransferrin or ferrilactoferrin in the acquisition of iron by promastigotes. Thus, L. chagasi promastigotes can acquire iron from hemin, ferrilactoferrin, or ferritransferrin. This capacity to utilize several iron sources may contribute to the organism's ability to survive in the diverse environments it encounters in the insect and mammalian hosts.