Proteomic analysis of Proteus mirabilis outer membrane proteins reveals differential expression in vivo vs. in vitro conditions

Proteus mirabilis is an opportunistic pathogen that frequently causes complicated urinary tract infections. Among a wide spectrum of potential virulence factors, outer membrane proteins (OMPs) are critical for bacterial interactions and survival in different environments. In this work, we used a proteomic approach to assess P. mirabilis in vivo OMPs expression compared to in vitro, including iron replete and iron-restricted conditions. Three putative iron receptors, IreA, PMI0842, and PMI2596, were detected both in bacterium grown in vivo and in vitro under iron-restricted conditions. A prophage gene product, PMI1721, was detected only on in vivo growing bacterium, suggesting a potential role yet to be disclosed on the surface of P. mirabilis. Plasminogen, a host protein, was co-purified with OMPs of in vivo grown bacteria, which is in accordance with previous observations and suggests that plasminogen bound to P. mirabilis surface may be associated to virulence as seen in other bacterial pathogens. Western blots using sera of experimentally challenged mice showed that iron-regulated proteins are expressed and highly immunogenic during infection. This work confirms observations made by others for P. mirabilis and reveals details not yet described, suggesting new aspects of the bacterium pathogenesis that remain unknown.     

Identification and characterization of SirA, an iron-regulated protein from Staphylococcus aureus

The acquisition of iron by pathogenic bacteria is often a crucial step in establishing infection. To accomplish this, many bacteria, including Staphylococcus aureus, produce low-molecular-weight iron-chelating siderophores. However, the secretion and transport of these molecules in gram-positive organisms are poorly understood. The sequence, organization, and regulation of genes involved in siderophore transport are conserved among gram-negative bacteria. We used this information to identify a putative siderophore transport locus from an S. aureus genomic sequence database. This locus contains three predicted open reading frames with a high degree of homology to genes involved in siderophore uptake in several bacterial species, in particular the cbr locus of the plant pathogen Erwinia chrysanthemi. The first gene in the locus, which we have designated sir for staphylococcal iron regulated, encodes a putative lipoprotein with a molecular mass of 37 kDa. The open reading frame is preceded by a 19-bp region of dyad symmetry with homology for operator sequences controlling iron-regulated expression of genes in other bacteria. Fur titration experiments indicate that this region of dyad symmetry is sufficient for Fur-dependent regulation in Escherichia coli. The expression of this gene was repressed, in a dose-dependent manner, by the addition of iron to the S. aureus culture medium. sir-encoded proteins may be involved in iron acquisition in vivo and therefore may be targets for antimicrobial agents.     

Quantitative Metabolomics Reveals an Epigenetic Blueprint for Iron Acquisition in Uropathogenic Escherichia coli

Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies.     

The Fe Chelator Proferrorosamine A Is Essential for the Siderophore-Mediated Uptake of Iron by Pseudomonas roseus fluorescens

Pseudomonas roseus fluorescens produces, besides the Fe chelator proferrorosamine A, Fe -chelating compounds, called siderophores. The production of proferrorosamine A and siderophores by P. roseus fluorescens appears to be controlled in a similar way by the concentration of available iron and by the concentration of dissolved oxygen. The higher the concentration of iron available for the microorganism, the lower the production of both chelating compounds. However, the production of siderophores was much more sensitive to iron availability than was proferrorosamine A production. Proferrorosamine A and siderophores were only produced in minimal medium C if the concentration of dissolved oxygen ranged from 4.5 to 2.0 ppm. At higher or lower concentrations, none of the iron-chelating compounds were produced. Furthermore, it has been shown that proferrorosamine-negative Tn5 mutants of P. roseus fluorescens were able to form siderophores only under iron-limiting conditions when proferrorosamine A was added to the medium. Our data suggest that proferrorosamine A production is essential for siderophore synthesis by P. roseus fluorescens; the production of siderophores occurred only when proferrorosamine A was present.     

Yersiniabactin and other siderophores produced by clinical isolates of Enterobacter spp. and Citrobacter spp

We analyzed the ability of extraintestinal strains of Enterobacter spp. and Citrobacter spp. to employ different siderophore-mediated strategies of iron acquisition. All strains produced iron-chelating compounds. Cross-feeding assays indicated that most isolates of both Enterobacter spp. and Citrobacter spp. excreted catecholate siderophore enterobactin, less produced aerobactin, and single strains excreted hydroxamates different from aerobactin. Besides, we analyzed if the strains had the ability to produce the siderophore yersiniabactin coded by the Yersinia high-pathogenicity island (HPI). The presence of HPI genes was observed in single isolates of three species: E. cloaceae, E. aerogenes and C. koseri. A detailed polymerase chain reaction analysis revealed differences in the genetic organization of the HPIs; however, in a cross-feeding test we proved that yersiniabactin was produced and the island was functional.     

Physiological properties and production of siderophores detected in Yersinia enterocolitica strain 4-32

A hydrophilic compound with siderophore activity was isolated from a culture of Yersinia enterocolitica 4-32 grown in an iron-deficient medium. It was found that the siderophore secreted did not belong to the catecholamide and hydroxamate type of siderophores and not yersiniabactin. Supplementation of cultures of Y. enterocolitica 4-32 with sodium chloride (300 mM) resulted in a decrease in the production of siderophores.     

The Fe2+ Chelator Proferrorosamine A Is Essential for the Siderophore-Mediated Uptake of Iron by Pseudomonas roseus fluorescens

Pseudomonas roseus fluorescens produces, besides the Fe²⁺ chelator proferrorosamine A, Fe³⁺ -chelating compounds, called siderophores. The production of proferrorosamine A and siderophores by P. roseus fluorescens appears to be controlled in a similar way by the concentration of available iron and by the concentration of dissolved oxygen. The higher the concentration of iron available for the microorganism, the lower the production of both chelating compounds. However, the production of siderophores was much more sensitive to iron availability than was proferrorosamine A production. Proferrorosamine A and siderophores were only produced in minimal medium C if the concentration of dissolved oxygen ranged from 4.5 to 2.0 ppm. At higher or lower concentrations, none of the iron-chelating compounds were produced. Furthermore, it has been shown that proferrorosamine-negative Tn5 mutants of P. roseus fluorescens were able to form siderophores only under iron-limiting conditions when proferrorosamine A was added to the medium. Our data suggest that proferrorosamine A production is essential for siderophore synthesis by P. roseus fluorescens; the production of siderophores occurred only when proferrorosamine A was present.     

Variation in Siderophore Biosynthetic Gene Distribution and Production across Environmental and Faecal Populations of Escherichia coli

Iron is essential for Escherichia coli growth and survival in the host and the external environment, but its availability is generally low due to the poor solubility of its ferric form in aqueous environments and the presence of iron-withholding proteins in the host. Most E. coli can increase access to iron by excreting siderophores such as enterobactin, which have a very strong affinity for Fe³⁺. A smaller proportion of isolates can generate up to 3 additional siderophores linked with pathogenesis; aerobactin, salmochelin, and yersiniabactin. However, non-pathogenic E. coli are also able to synthesise these virulence-associated siderophores. This raises questions about their role in the ecology of E. coli, beyond virulence, and whether specific siderophores might be linked with persistence in the external environment. Under the assumption that selection favours phenotypes that confer a fitness advantage, we compared siderophore production and gene distribution in E. coli isolated either from agricultural plants or the faeces of healthy mammals. This population-level comparison has revealed that under iron limiting growth conditions plant-associated isolates produced lower amounts of siderophores than faecal isolates. Additionally, multiplex PCR showed that environmental isolates were less likely to contain loci associated with aerobactin and yersiniabactin synthesis. Although aerobactin was linked with strong siderophore excretion, a significant difference in production was still observed between plant and faecal isolates when the analysis was restricted to strains only able to synthesise enterobactin. This finding suggests that the regulatory response to iron limitation may be an important trait associated with adaptation to the non-host environment. Our findings are consistent with the hypothesis that the ability to produce multiple siderophores facilitates E. coli gut colonisation and plays an important role in E. coli commensalism.     

Phage selection for bacterial cheats leads to population decline

While predators and parasites are known for their effects on bacterial population biology, their impact on the dynamics of bacterial social evolution remains largely unclear. Siderophores are iron-chelating molecules that are key to the survival of certain bacterial species in iron-limited environments, but their production can be subject to cheating by non-producing genotypes. In a selection experiment conducted over approximately 20 bacterial generations and involving 140 populations of the pathogenic bacterium Pseudomonas aeruginosa PAO1, we assessed the impact of a lytic phage on competition between siderophore producers and non-producers. We show that the presence of lytic phages favours the non-producing genotype in competition, regardless of whether iron use relies on siderophores. Interestingly, phage pressure resulted in higher siderophore production, which constitutes a cost to the producers and may explain why they were outcompeted by non-producers. By the end of the experiment, however, cheating load reduced the fitness of mixed populations relative to producer monocultures, and only monocultures of producers managed to grow in the presence of phage in situations where siderophores were necessary to access iron. These results suggest that public goods production may be modulated in the presence of natural enemies with consequences for the evolution of social strategies.     

An overview of siderophores for iron acquisition in microorganisms living in the extreme

Siderophores are iron-chelating molecules produced by microbes when intracellular iron concentrations are low. Low iron triggers a cascade of gene activation, allowing the cell to survive due to the synthesis of important proteins involved in siderophore synthesis and transport. Generally, siderophores are classified by their functional groups as catecholates, hydroxamates and hydroxycarboxylates. Although other chemical structural modifications and functional groups can be found. The functional groups participate in the iron-chelating process when the ferri-siderophore complex is formed. Classified as acidophiles, alkaliphiles, halophiles, thermophiles, psychrophiles, piezophiles, extremophiles have particular iron requirements depending on the environmental conditions in where they grow. Most of the work done in siderophore production by extremophiles is based in siderophore concentration and/or genomic studies determining the presence of siderophore synthesis and transport genes. Siderophores produced by extremophiles are not well known and more work needs to be done to elucidate chemical structures and their role in microorganism survival and metal cycling in extreme environments.     

The mineral weathering ability of Collimonas pratensis PMB3(1) involves a Malleobactin-mediated iron acquisition system

Mineral weathering by microorganisms is considered to occur through a succession of mechanisms based on acidification and chelation. While the role of acidification is established, the role of siderophores is difficult to disentangle from the effect of the acidification. We took advantage of the ability of strain Collimonas pratensis PMB3(1) to weather minerals but not to acidify depending on the carbon source to address the role of siderophores in mineral weathering. We identified a single non-ribosomal peptide synthetase (NRPS) responsible for siderophore biosynthesis in the PMB3(1) genome. By combining iron-chelating assays, targeted mutagenesis and chemical analyses (HPLC and LC-ESI-HRMS), we identified the siderophore produced as malleobactin X and how its production depends on the concentration of available iron. Comparison with the genome sequences of other collimonads evidenced that malleobactin production seems to be a relatively conserved functional trait, though some collimonads harboured other siderophore synthesis systems. We also revealed by comparing the wild-type strain and its mutant impaired in the production of malleobactin that the ability to produce this siderophore is essential to allow the dissolution of hematite under non-acidifying conditions. This study represents the first characterization of the siderophore produced by collimonads and its role in mineral weathering.     

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.     

Petrobactin,a siderophore produced by Alteromonas,mediates community iron acquisition in the global ocean

It is now widely accepted that siderophores play a role in marine iron biogeochemical cycling. However, the mechanisms by which siderophores affect the availability of iron from specific sources and the resulting significance of these processes on iron biogeochemical cycling as a whole have remained largely untested. In this study, we develop a model system for testing the effects of siderophore production on iron bioavailability using the marine copiotroph Alteromonas macleodii ATCC 27126. Through the generation of the knockout cell line ΔasbB::km^r, which lacks siderophore biosynthetic capabilities, we demonstrate that the production of the siderophore petrobactin enables the acquisition of iron from mineral sources and weaker iron-ligand complexes. Notably, the utilization of lithogenic iron, such as that from atmospheric dust, indicates a significant role for siderophores in the incorporation of new iron into marine systems. We have also detected petrobactin, a photoreactive siderophore, directly from seawater in the mid-latitudes of the North Pacific and have identified the biosynthetic pathway for petrobactin in bacterial metagenome-assembled genomes widely distributed across the global ocean. Together, these results improve our mechanistic understanding of the role of siderophore production in iron biogeochemical cycling in the marine environment wherein iron speciation, bioavailability, and residence time can be directly influenced by microbial activities.Subject terms: Biogeochemistry, Marine microbiology     

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.     

Yersinia high pathogenicity island genes modify the Escherichia coli primary metabolome independently of siderophore production

Bacterial siderophores may enhance pathogenicity by scavenging iron, but their expression has been proposed to exert a substantial metabolic cost. Here we describe a combined metabolomic-genetic approach to determine how mutations affecting the virulence-associated siderophore yersiniabactin affect the Escherichia coli primary metabolome. Contrary to expectations, we did not find yersiniabactin biosynthesis to correspond to consistent metabolomic shifts. Instead, we found that targeted deletion of ybtU or ybtA, dissimilar genes with similar roles in regulating yersiniabactin expression, were associated with a specific shift in arginine pathway metabolites during growth in minimal media. This interaction was associated with high arginine levels in the model uropathogen Escherichia coli UTI89 compared to its ybtU and ybtA mutants and the K12 strain MG1655, which lacks yersiniabactin-associated genes. Because arginine is not a direct yersiniabactin biosynthetic substrate, these findings show that virulence-associated secondary metabolite systems may shape bacterial primary metabolism independently of substrate consumption.     

Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA

One alternative method for drug delivery involves the use of siderophore-antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron-chelating siderophores, can be actively transported across the outer membrane of gram-negative bacteria. These "Trojan Horse" antibiotics may prove useful as an efficient means to combat multi-drug-resistant bacterial infections. Here we present the crystallographic structures of the natural siderophore-antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad-host range antibiotics that consist of a hydroxamate-type iron-chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate-type siderophores, the three-dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron-chelating siderophores and offers a structural platform for the rational design of hydroxamate-type siderophore-antibiotic conjugates.