Siderophore System Research Articles

Multifaceted regulation of siderophore synthesis by multiple regulatory systems in Shewanella oneidensis

Siderophore-dependent iron uptake is a mechanism by which microorganisms scavenge and utilize iron for their survival, growth, and many specialized activities, such as pathogenicity. The siderophore biosynthetic system PubABC in Shewanella can synthesize a series of distinct siderophores, yet how it is regulated in response to iron availability remains largely unexplored. Here, by whole genome screening we identify TCS components histidine kinase (HK) BarA and response regulator (RR) SsoR as positive regulators of siderophore biosynthesis. While BarA partners with UvrY to mediate expression of pubABC post-transcriptionally via the Csr regulatory cascade, SsoR is an atypical orphan RR of the OmpR/PhoB subfamily that activates transcription in a phosphorylation-independent manner. By combining structural analysis and molecular dynamics simulations, we observe conformational changes in OmpR/PhoB-like RRs that illustrate the impact of phosphorylation on dynamic properties, and that SsoR is locked in the ‘phosphorylated’ state found in phosphorylation-dependent counterparts of the same subfamily. Furthermore, we show that iron homeostasis global regulator Fur, in addition to mediating transcription of its own regulon, acts as the sensor of iron starvation to increase SsoR production when needed. Overall, this study delineates an intricate, multi-tiered transcriptional and post-transcriptional regulatory network that governs siderophore biosynthesis.

Yersiniabactin is a quorum-sensing autoinducer and siderophore in uropathogenic Escherichia coli.

Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all Escherichia coli express the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally express the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared the growth of different isogenic siderophore biosynthetic mutants in the presence of transferrin, a human iron-binding protein. We observed that Ybt expression does not compensate for deficient Ent expression following low-density inoculation. Using transcriptional and product analysis, we found this non-redundancy to be attributable to a density-dependent transcriptional stimulation cycle in which Ybt functions as an autoinducer. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. This combined functionality may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales.IMPORTANCEPatients with urinary tract infections are often infected with Escherichia coli strains carrying adaptations that increase their pathogenic potential. One of these adaptations is the accumulation of multiple siderophore systems, which scavenge iron for nutritional use. While iron uptake is important for bacterial growth, the increased metabolic costs of siderophore production could diminish bacterial fitness during infections. In a siderophore-dependent growth condition, we show that the virulence-associated yersiniabactin siderophore system in uropathogenic E. coli is not redundant with the ubiquitous E. coli enterobactin system. This arises not from differences in iron-scavenging activity but because yersiniabactin is preferentially expressed during bacterial crowding, leaving bacteria dependent upon enterobactin for growth at low cell density. Notably, this regulatory mode arises because yersiniabactin stimulates its own expression, acting as an autoinducer in a previously unappreciated quorum-sensing system. This unexpected result connects quorum-sensing with pathogenic potential in E. coli and related Enterobacterales.

The phylogenomic landscape of extended-spectrum β-lactamase producing Citrobacter species isolated from surface water

BackgroundCitrobacter species are Gram-negative opportunistic pathogens commonly reported in nosocomial-acquired infections. This study characterised four Citrobacter species that were isolated from surface water in the North West Province, South Africa.ResultsPhenotypic antimicrobial susceptibility profiles of the isolates demonstrated their ability to produce the extended-spectrum β-lactamase (ESBL). Whole genomes were sequenced to profile antibiotic resistance and virulence genes, as well as mobile genetic elements. In silico taxonomic identification was conducted by using multi-locus sequence typing and average nucleotide identity. A pangenome was used to determine the phylogenomic landscape of the Citrobacter species by using 109 publicly available genomes. The strains S21 and S23 were identified as C. braakii, while strains S24 and S25 were C. murliniae and C. portucalensis, respectively. Comparative genomics and sequenced genomes of the ESBL-producing isolates consisted of n = 91; 83% Citrobacter species in which bla-CMY−101 (n = 19; 32,2%) and bla-CMY−59 (n = 12; 38,7%) were prevalent in C. braakii, and C. portucalensis strains, respectively. Macrolide (acrAB-TolC, and mdtG) and aminoglycoside (acrD) efflux pumps genes were identified in the four sequenced Citrobacter spp. isolates. The quinolone resistance gene, qnrB13, was exclusive to the C. portucalensis S25 strain. In silico analysis detected plasmid replicon types IncHI1A, IncP, and Col(VCM04) in C. murliniae S24 and C. portucalensis S25, respectively. These potentially facilitate the T4SS secretion system in Citrobacter species. In this study, the C. braakii genomes could be distinguished from C. murliniae and C. portucalensis on the basis of gene encoding for cell surface localisation of the CPS (vexC) and identification of genes involved in capsule polymer synthesis (tviB and tviE). A cluster for the salmochelin siderophore system (iro-BCDEN) was found in C. murliniae S24. This is important when it comes to the pathogenicity pathway that confers an advantage in colonisation.ConclusionsThe emerging and genomic landscapes of these ESBL-producing Citrobacter species are of significant concern due to their dissemination potential in freshwater systems. The presence of these ESBL and multidrug-resistant (MDR) pathogens in aquatic environments is of One Health importance, since they potentially impact the clinical domain, that is, in terms of human health and the agricultural domain, that is, in terms of animal health and food production as well as the environmental domain.

Fungal siderophore metabolism with a focus on Aspergillus fumigatus: impact on biotic interactions and potential translational applications.

Iron is an essential trace element that is limiting in most habitats including hosts for fungal pathogens. Siderophores are iron-chelators synthesized by most fungal species for high-affinity uptake and intracellular handling of iron. Moreover, virtually all fungal species including those lacking siderophore biosynthesis appear to be able to utilize siderophores produced by other species. Siderophore biosynthesis has been shown to be crucial for virulence of several fungal pathogens infecting animals and plants revealing induction of this iron acquisition system during virulence, which offers translational potential of this fungal-specific system. The present article summarizes the current knowledge on the fungal siderophore system with a focus on Aspergillus fumigatus and its potential translational application including noninvasive diagnosis of fungal infections via urine samples, imaging of fungal infections via labeling of siderophores with radionuclides such as Gallium-68 for detection with positron emission tomography, conjugation of siderophores with fluorescent probes, and development of novel antifungal strategies.

The transmutation of Escherichia coli ATCC 25922 to small colony variants (SCVs) E. coli strain as a result of exposure to gentamicin

BackgroundSmall colony variants (SCVs) are biotypes of bacteria that have a size of approximately one-tenth or less of the wild types and has distinct characteristics comparing to the wild type strains. Clinical SCVs are usually associated with persistent infection and require a long-term treatment program with antibiotics. In Saudi Arabia, there are few studies about SCVs Escherichia coli for this reason, this study is aimed to investigate the ability of gentamicin to mutate E. coli ATCC 25922 to produce small SCVs and investigate the genotypes and phenotypes changes and stress tolerance comparing to clinical SCVs E. coli and normal clinical E. coli Isolated from blood samples. MethodsIn this investigation, four clinical blood samples were collected ted from patients and the cultivation and isolation were carried out in KFMC between December 2019 and February 2021. The identification of positive blood culture samples was done using phoenix MD. Non-SCV E. coli ATCC25922 were mutated to SCV using exposure to increasing gradual concentrations of gentamicin at 100-generation intervals. Biochemical features and susceptibility to standard antibiotics using automated Phoenix MD 50 and. The survival assays were done using several stresses including heat shock, low pH, high osmotic pressure, and oxidative pressure. Virulence genes screening included the detection of genes that encoded to α-haemolysin, CS12 fimbriae, F17-like fimbrial adhesion, P-related fimbriae, yersiniabactin siderophore system, P-fimbriae, aerobactin, iron-regulated genes using PCR and gel electrophoresis. ResultsThe data from the mutating E. coli ATCC 25922 small colony test with gentamicin revealed that the first emergence of the multidrug resistance (MDR) SCV E. coli strain occurred at generation number 250, corresponding to a gentamicin concentration of 57 g/ml. Pathogenicity islands detection revealed that all tested E. coli strains have PAI IV 536 genes on their chromosomes furthermore, mutated SCV E. coli ATCC 25922 acquired PAII CFT073 and PAI IV 536. Survival tests showed no significant differences changes in tolerance of mutated SCVs comparing to parental strain. ConclusionThe present work concluded that gentamicin sub-MIC concentration gradual exposure can induce mutation responsible for SCV formation and evolving of MDR E. coli strains. The mutated SCVs evolved high-level aminoglycoside resistance for gentamicin and resistance to amikacin, it also developed resistance to 2 cephalosporin antibiotics cefuroxime, and cephalothin and a resistance to aztreonam.

Mycobacterium abscessus Mutants with a Compromised Functional Link between the Type VII ESX-3 System and an Iron Uptake Mechanism Reliant on an Unusual Mycobactin Siderophore

The opportunistic pathogen Mycobacterium abscessus subsp. abscessus (Mab) has become an emerging public health threat due to the increasing number of Mab-associated chronic pulmonary disease cases. Treatment requires multiple drug courses and is often combined with surgical resection. Cure rates are only ~50% due to treatment failure and comorbidities. Deeper understanding of the biology of Mab is required to illuminate potential avenues for the development of better therapeutics against Mab infections. The ESX-3 type VII protein secretion system of Mab has an important role in host inflammatory and pathological responses during infection. In this work, we demonstrate a functional link between ESX-3 and an iron uptake system based on an unusual mycobactin-type siderophore (designated MBT Ab) and exploit this link to implement a large screen for transposon mutants with an impaired ESX-3. Most mutants we identified carry insertions in genes encoding predicted ESX-3 secretion machinery components or potential ESX-3 substrates. The mutants overproduce MBT Ab, a trait consistent with an iron uptake defect. Our characterization of MBT Ab revealed structural features reminiscent of nocardial mycobactin-like compounds with cytotoxicity. This finding raises the possibility that MBT Ab may play roles in pathogenesis unlinked to iron homeostasis. The mutants generated herein will facilitate research to better understand the role of ESX-3 and its interplay with the siderophore system.

Identification of Key Functions Required for Production and Utilization of the Siderophore Piscibactin Encoded by the High-Pathogenicity Island irp-HPI in Vibrionaceae.

Piscibactin is a widespread siderophore system present in many different bacteria, especially within the Vibrionaceae family. Previous works showed that most functions required for biosynthesis and transport of this siderophore are encoded by the high-pathogenicity island irp-HPI. In the present work, using Vibrio anguillarum as a model, we could identify additional key functions encoded by irp-HPI that are necessary for piscibactin production and transport and that have remained unknown. Allelic exchange mutagenesis, combined with cross-feeding bioassays and LC-MS analysis, were used to demonstrate that Irp4 protein is an essential component for piscibactin synthesis since it is the thioesterase required for nascent piscibactin be released from the NRPS Irp1. We also show that Irp8 is a MFS-type protein essential for piscibactin secretion. In addition, after passage through the outer membrane transporter FrpA, the completion of ferri-piscibactin internalization through the inner membrane would be achieved by the ABC-type transporter FrpBC. The expression of this transporter is coordinated with the expression of FrpA and with the genes encoding biosynthetic functions. Since piscibactin is a major virulence factor of some pathogenic vibrios, the elements of biosynthesis and transport described here could be additional interesting targets for the design of novel antimicrobials against these bacterial pathogens.

Population snapshot of the extended-spectrum \u03b2-lactamase-producing Escherichia coli invasive strains isolated from a Hungarian hospital

BackgroundThis study was carried out to determine the prevalence and the genetic background of extended-spectrum β-lactamase-producing Escherichia coli invasive isolates obtained from a tertiary-care hospital in Budapest, Hungary.MethodsBetween October–November 2018, all invasive ESBL-producing E. coli isolates were collected from Central Hospital of Southern Pest. The antimicrobial susceptibility testing was performed according to the EUCAST guidelines. The possible clonal relationships were investigated by core genome (cg)MLST (SeqSphere +) using whole-genome sequencing (WGS) data of isolates obtained from Illumina 251-bp paired-end sequencing. From WGS data acquired antimicrobial resistance genes, virulence genes and replicon types were retrieved using ResFinder3.1, PlasmidFinder2.1, pMLST-2.0, VirulenceFinder2.0 and Virulence Factors Database online tools.ResultsOverall, six E. coli isolates proved to be resistant to third-generation cephalosporins and ESBL-producers in the study period. Full genome sequence analysis showed that five E. coli isolates belonged to the ST131 clone: two to C1-M27 subclade with blaCTX-M-27 and three to C2/H30Rx subclade with blaCTX-M-15. One isolate belonged to ST1193 with blaCTX-M-27. According to cgMLST, all C2/H30Rx isolates formed a cluster (≤ 6 allele differences), while the blaCTX-M-27-producing C1-M27 isolates differed at least 35 alleles from each other. Both C2/H30Rx and C1-M27 ST131 isolates harbored similar antimicrobial resistance gene sets. However, only C2/H30Rx isolates had the qnrB and aac(3)-IIa. The isolates carried similar extraintestinal virulence gene set but differed in some genes encoding siderophores, protectins and toxins. Moreover, only one C2/H30Rx isolate carried salmochelin siderophore system and showed virotype B. All isolates showed resistance against ceftriaxone, cefotaxime, and ciprofloxacin, and the C2/H30Rx isolates were also resistant to gentamicin, tobramycin, and ceftazidime.ConclusionsOut of six ESBL-producing E. coli, five belonged to the ST131 clone. This study indicates, that the C2/H30Rx and C1-M27 subclades of the ST131 appear to be the dominant clones collected in a Hungarian hospital.

The Temperature-Dependent Expression of the High-Pathogenicity Island Encoding Piscibactin in Vibrionaceae Results From the Combined Effect of the AraC-Like Transcriptional Activator PbtA and Regulatory Factors From the Recipient Genome.

The high-pathogenicity island irp-HPI is widespread among Vibrionaceae encoding the piscibactin siderophore system. The expression of piscibactin genes in the fish pathogen Vibrio anguillarum is favored by low temperatures. However, information about the regulatory mechanism behind irp-HPI gene expression is scarce. In this work, in-frame deletion mutants of V. anguillarum defective in the putative regulators AraC1 and AraC2, encoded by irp-HPI, and in the global regulators H-NS and ToxRS, were constructed and their effect on irp-HPI gene expression was analyzed at 15 and 25°C. The results proved that only AraC1 (renamed as PbtA) is required for the expression of piscibactin biosynthesis and transport genes. PbtA inactivation led to an inability to grow under iron restriction, a loss of the outer membrane piscibactin transporter FrpA, and a significant decrease in virulence for fish. Inactivation of the global repressor H-NS, which is involved in silencing of horizontally acquired genes, also resulted in a lower transcriptional activity of the frpA promoter. Deletion of toxR-S, however, did not have a relevant effect on the expression of the irp-HPI genes. Therefore, while irp-HPI would not be part of the ToxR regulon, H-NS must exert an indirect effect on piscibactin gene expression. Thus, the temperature-dependent expression of the piscibactin-encoding pathogenicity island described in V. anguillarum is the result of the combined effect of the AraC-like transcriptional activator PbtA, harbored in the island, and other not yet defined regulator(s) encoded by the genome. Furthermore, different expression patterns were detected within different irp-HPI evolutionary lineages, which supports a long-term evolution of the irp-HPI genomic island within Vibrionaceae. The mechanism that modulates piscibactin gene expression could also be involved in global regulation of virulence factors in response to temperature changes.

Vibrio neptunius Produces Piscibactin and Amphibactin and Both Siderophores Contribute Significantly to Virulence for Clams.

Vibrio neptunius is an inhabitant of mollusc microbiota and an opportunistic pathogen causing disease outbreaks in marine bivalve mollusc species including oysters and clams. Virulence of mollusc pathogenic vibrios is mainly associated with the production of extracellular products. However, siderophore production is a common feature in pathogenic marine bacteria but its role in fitness and virulence of mollusc pathogens remains unknown. We previously found that V. neptunius produces amphibactin, one of the most abundant siderophores in marine microbes. In this work, synthesis of the siderophore piscibactin was identified as the second siderophore produced by V. neptunius. Single and double mutants in biosynthetic genes of each siderophore system, piscibactin and amphibactin, were constructed in V. neptunius and their role in growth ability and virulence was characterized. Although the High Pathogenicity Island encoding piscibactin is a major virulence factor in vibrios pathogenic for fish, the V. neptunius wild type did not cause mortality in turbot. The results showed that amphibactin contributes more than piscibactin to bacterial fitness in vitro. However, infection challenges showed that each siderophore system contributes equally to virulence for molluscs. The V. neptunius strain unable to produce any siderophore was severely impaired to cause vibriosis in clams. Although the inactivation of one of the two siderophore systems (either amphibactin or piscibactin) significantly reduced virulence compared to the wild type strain, the ability to produce both siderophores simultaneously maximised the degree of virulence. Evaluation of the gene expression pattern of each siderophore system showed that they are simultaneously expressed when V. neptunius is cultivated under low iron availability in vitro and ex vivo. Finally, the analysis of the distribution of siderophore systems in genomes of Vibrio spp. pathogenic for molluscs showed that the gene clusters encoding amphibactin and piscibactin are widespread in the Coralliilyticus clade. Thus, siderophore production would constitute a key virulence factor for bivalve molluscs pathogenic vibrios.

Aerobactin-Mediated Iron Acquisition Enhances Biofilm Formation, Oxidative Stress Resistance, and Virulence of Yersinia pseudotuberculosis.

Aerobactin is a citrate-hydroxamate siderophore that is critical for the virulence of pathogenic enteric bacteria. However, although the aerobactin-producing iucABCD-iutA operon is distributed widely in the genomes of Yersinia species, none of the pathogenic Yersinia spp. was found to produce aerobactin. Here, we showed that the iucABCD-iutA operon in the food-borne enteric pathogen Yersinia pseudotuberculosis YPIII is a functional siderophore system involved in iron acquisition. The expression of the operon was found to be directly repressed by the ferric uptake regulator (Fur) in an iron concentration-dependent manner. In addition, we demonstrated that the aerobactin-mediated iron acquisition contributes to bacterial growth under iron-limited conditions. Moreover, we provided evidence that aerobactin plays important roles in biofilm formation, resistance to oxidative stress, ROS removal, and virulence of Y. pseudotuberculosis. Overall, our study not only uncovered a novel strategy of iron acquisition in Y. pseudotuberculosis but also highlighted the importance of aerobactin in the pathogenesis of Y. pseudotuberculosis.

Leveraging copper import by yersiniabactin siderophore system for targeted PET imaging of bacteria.

There is an emerging need for accurate and rapid identification of bacteria in the human body to achieve diverse biomedical objectives. Copper homeostasis is vital for the survival of bacterial species owing to the roles of the metal as a nutrient, respiratory enzyme cofactor, and a toxin. Here, we report the development of a copper-64–labeled bacterial metal chelator, yersiniabactin, to exploit a highly conserved metal acquisition pathway for noninvasive and selective imaging of bacteria. Compared with traditional techniques used to manufacture probes, our strategy simplifies the process considerably by combining the function of metal attachment and cell recognition to the same molecule. We demonstrate, for the first time to our knowledge, how a copper-64 PET probe can be used to identify specific bacterial populations, monitor antibiotic treatment outcomes, and track bacteria in diverse niches in vivo.

Proteomic analysis deciphers the multi-targeting antivirulence activity of tannic acid in modulating the expression of MrpA, FlhD, UreR, HpmA and Nrp system in Proteus mirabilis

In the present study, the multi-targeting antivirulence activity of tannic acid (TA) was explored against Proteus mirabilis through MS-based proteomic approach. The in vitro biofilm biomass quantification assay and microscopic analysis demonstrated the antibiofilm activity of TA against P. mirabilis in which, minimum biofilm inhibitory concentration (MBIC) of TA was found to be 200 μg/mL concentration. Moreover, the nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) analysis revealed that TA (at MBIC) differentially regulated the proteins involved in fimbrial adhesion, flagellar motility, iron acquisition, Fe-S cluster assembly, heat shock response, virulence enzymes, and toxin secretion. Further, the transcriptomic analysis validated the outcomes of proteomic analysis in which, the expression level of virulence genes responsible for MR/P fimbrial adhesion (mrpA), flagellar transcriptional activation (flhD), biosynthesis of urease (ureR), hemolysin (hpmA), non-ribosomal peptide siderophore system (Nrp), oxidative stress responsible enzymes and fitness factors proteins were down-regulated in TA exposed P. mirabilis. These observations were also in correspondence with the in vitro bioassays. Thus, this study reports the feasibility of TA to act as a promising therapeutic agent against multifactorial P. mirabilis infections.

Alternaria alternata uses two siderophore systems for iron acquisition

Iron is one of the most abundant elements on earth and essential for life. However, Fe3+ ions are rather insoluble and microorganisms such as fungi may use siderophores as strong chelators for uptake. In addition, free cytoplasmic iron is rather toxic and intracellular siderophores are used to control the toxicity. Siderophores are also important for iron storage. We studied two siderophore systems in the plant necrotrophic fungus Alternaria alternata and show that the non-ribosomal peptide synthase, Nps2, is required for the biosynthesis of intracellular ferricrocin, whereas Nps6 is needed for the formation of extracellular coprogen and coprogen B. Whereas nps2 was dispensable for growth on iron-depleted medium, nps6 was essential under those conditions. nps2 deletion caused an increase in spore formation and reduced pathogenicity on tomato. Our results suggest that A. alternata employs an external and an internal siderophore system to adapt to low iron conditions.

The Expression of Virulence Factors in Vibrio anguillarum Is Dually Regulated by Iron Levels and Temperature.

Vibrio anguillarum causes a hemorrhagic septicemia that affects cold- and warm-water adapted fish species. The main goal of this work was to determine the temperature-dependent changes in the virulence factors that could explain the virulence properties of V. anguillarum for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25°C, the degree of virulence of V. anguillarum RV22 is higher at 15°C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of V. anguillarum RV22 cultured under low-iron availability at either 25 or 15°C, which would mimic the conditions that V. anguillarum finds during colonization of fish cultivated at warm- or cold-water temperatures. The comparative analysis of transcriptomes at high- and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25°C than at 15°C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15°C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25°C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that V. anguillarum adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow V. anguillarum to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.

Contribution of yersiniabactin to the virulence of an Escherichia coli sequence type 69 (“clonal group A”) cystitis isolate in murine models of urinary tract infection and sepsis

Escherichia coli sequence type 69 (ST69; “clonal group A”) is an important extraintestinal pathogen. To clarify the yersiniabactin siderophore system's role in ST69's extraintestinal virulence we compared a wild-type ST69 cystitis isolate, isogenic irp2 (yersiniabactin) mutants, and irp2-complemented mutants in murine models of sepsis and urinary tract infection (UTI). irp2 mutants were attenuated mildly in the UTI model and profoundly in the sepsis model. In both models, complementation with a functional copy of irp2 restored full parental virulence. These findings suggest that in ST69 the yersiniabactin system has a minor role in urovirulence and a major role in sepsis causation.

Stenotrophomonas maltophilia produces an EntC-dependent catecholate siderophore that is distinct from enterobactin.

Stenotrophomonas maltophilia, a Gram-negative, multi-drug-resistant bacterium, is increasingly recognized as a key opportunistic pathogen. Thus, we embarked upon an investigation of S. maltophilia iron acquisition. To begin, we determined that the genome of strain K279a is predicted to encode a complete siderophore system, including a biosynthesis pathway, an outer-membrane receptor for ferrisiderophore, and other import and export machinery. Compatible with these data, K279a and other clinical isolates of S. maltophilia secreted a siderophore-like activity when grown at 25-37 °C in low-iron media, as demonstrated by a chrome azurol S assay, which detects iron chelation, and Arnow and Rioux assays, which detect catecholate structures. Importantly, these supernatants rescued the growth of iron-starved S. maltophilia, documenting the presence of a biologically active siderophore. A mutation in one of the predicted biosynthesis genes (entC) abolished production of the siderophore and impaired bacterial growth in low-iron conditions. Inactivation of the putative receptor gene (fepA) prevented the utilization of siderophore-containing supernatants for growth in low-iron conditions. Although the biosynthesis and import loci showed some similarity to those of enterobactin, a well-known catecholate made by enteric bacteria, the siderophore of K279a was unable to rescue the growth of an enterobactin-utilizing indicator strain, and conversely iron-starved S. maltophilia could not use purified enterobactin. Furthermore, the S. maltophilia siderophore displayed patterns of solubility in organic compounds and mobility upon thin-layer chromatography that were distinct from those of enterobactin and its derivative, salmochelin. Together, these data demonstrate that S. maltophilia secretes a novel catecholate siderophore.

Enterobacteria secrete an inhibitor of Pseudomonas virulence during clinical bacteriuria.

Escherichia coli and other Enterobacteriaceae are among the most common pathogens of the human urinary tract. Among the genetic gains of function associated with urinary E. coli isolates is the Yersinia high pathogenicity island (HPI), which directs the biosynthesis of yersiniabactin (Ybt), a virulence-associated metallophore. Using a metabolomics approach, we found that E. coli and other Enterobacteriaceae expressing the Yersinia HPI also secrete escherichelin, a second metallophore whose chemical structure matches a known synthetic inhibitor of the virulence-associated pyochelin siderophore system in Pseudomonas aeruginosa. We detected escherichelin during clinical E. coli urinary tract infection (UTI) and experimental human colonization with a commensal, potentially probiotic E. coli bacteriuria strain. Escherichelin production by colonizing enterobacteria may help human hosts resist opportunistic infections by Pseudomonas and other pyochelin-expressing bacteria. This siderophore-based mechanism of microbial antagonism may be one of many elements contributing to the protective effects of the human microbiome. Future UTI-preventive probiotic strains may benefit by retaining the escherichelin biosynthetic capacity of the Yersinia HPI while eliminating the Ybt biosynthetic capacity.

Secreted Citrate Serves as Iron Carrier for the Marine Pathogen Photobacterium damselae subsp damselae.

Photobacterium damselae subsp damselae (Pdd) is a Vibrionaceae that has a wide pathogenic potential against many marine animals and also against humans. Some strains of this bacterium acquire iron through the siderophore vibrioferrin. However, there are virulent strains that do not produce vibrioferrin, but they still give a strong positive reaction in the CAS test for siderophore production. In an in silico search on the genome sequences of this type of strains we could not find any ORF which could be related to a siderophore system. To identify genes that could encode a siderophore-mediated iron acquisition system we used a mini-Tn10 transposon random mutagenesis approach. From more than 1,400 mutants examined, we could isolate a mutant (BP53) that showed a strong CAS reaction independently of the iron levels of the medium. In this mutant the transposon was inserted into the idh gene, which encodes an isocitrate dehydrogenase that participates in the tricarboxylic acid cycle. The mutant did not show any growth impairment in rich or minimal media, but it accumulated a noticeable amount of citrate (around 7 mM) in the culture medium, irrespective of the iron levels. The parental strain accumulated citrate, but in an iron-regulated fashion, being citrate levels 5–6 times higher under iron restricted conditions. In addition, a null mutant deficient in citrate synthase showed an impairment for growth at high concentrations of iron chelators, and showed almost no reaction in the CAS test. Chemical analysis by liquid chromatography of the iron-restricted culture supernatants resulted in a CAS-positive fraction with biological activity as siderophore. HPLC purification of that fraction yielded a pure compound which was identified as citrate from its MS and NMR spectral data. Although the production of another citrate-based compound with siderophore activity cannot be ruled out, our results suggest that Pdd secretes endogenous citrate and use it for iron scavenging from the cell environment.

Flying under the radar: The non-canonical biochemistry and molecular biology of petrobactin from Bacillus anthracis.

The dramatic, rapid growth of Bacillus anthracis that occurs during systemic anthrax implies a crucial requirement for the efficient acquisition of iron. While recent advances in our understanding of B. anthracis iron acquisition systems indicate the use of strategies similar to other pathogens, this review focuses on unique features of the major siderophore system, petrobactin. Ways that petrobactin differs from other siderophores include: A. unique ferric iron binding moieties that allow petrobactin to evade host immune proteins; B. a biosynthetic operon that encodes enzymes from both major siderophore biosynthesis classes; C. redundancy in membrane transport systems for acquisition of Fe-petrobactin holo-complexes; and, D. regulation that appears to be controlled predominately by sensing the host-like environmental signals of temperature, CO2 levels and oxidative stress, as opposed to canonical sensing of intracellular iron levels. We argue that these differences contribute in meaningful ways to B. anthracis pathogenesis. This review will also outline current major gaps in our understanding of the petrobactin iron acquisition system, some projected means for exploiting current knowledge, and potential future research directions.