Analysis of Microbial Siderophores by Mass Spectrometry.

Siderophores represent important microbial virulence factors and infection biomarkers. Their monitoring in fermentation broths, bodily fluids, and tissues should be reproducible. Similar isolation, characterization, and quantitation studies can often have conflicting results, and without proper documentation of sample collection, data processing, and analysis methods, it is difficult to reexamine the data and reconcile these differences. In this Springer Nature Protocol, we present the procedure optimized for ferricrocin/triacetylfusarinine C extraction from biological material as well as for tissue fixation and cryosectioning for optical microscopy and for both elemental and molecular mass spectrometry imaging. Special attention is paid to siderophore data mining from conventional and product ion mass spectra, liquid chromatography, and mass spectrometry imaging datasets, performed here by our free software called CycloBranch.

Ferrichrome identified from Lactobacillus casei ATCC334 induces apoptosis through its iron-binding site in gastric cancer cells.

Ferrichrome is known to be a siderophore, but it was recently identified as a tumor-suppressive molecule derived from Lactobacillus casei ATCC334 ( L. casei). In the present study, we investigated the effects of ferrichrome in gastric cancer cells. Cell lines and xenograft models treated with ferrichrome demonstrated growth suppression. The expression levels of cleaved poly (adenosine diphosphate-ribose) polymerase, and cleaved caspase-9 were increased by ferrichrome treatment. Although the tumor-suppressive effects of ferrichrome were almost completely diminished by the iron chelation, the reduction in the intracellular iron by ferrichrome did not correlate with its tumor-suppressive effects. An exhaustive docking simulation indicated that iron-free ferrichrome can make stable conformations with various mammalian molecules, including transporters and receptors. In conclusion, probiotic-derived ferrichrome induced apoptosis in gastric cancer cells. The iron binding site of ferrichrome is the structure responsible for its tumor suppressive function.

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor.

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H](+)). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C33H52N6O13Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.

[A IL-6R antagonist 2520A produced by a fungal species].

AIM: To isolate IL-6R antagonists from the cultured broth of the strain Torulomyces ovatus. METHODS: Various column chromatographyes were used to separate and purify the compounds with IL-6R antagonist activity. The spectral data and physic-chemical properties were measured for structure identification. RESULTS: One compound namely 2520 was isolated from the cultured broth of Torulomyces ovatus. CONCLUSION: 2520A is a known compound (ferrichrome). It is first reported about its antagonistic activity of IL-6R and identification of iron atom in its structure.

Ferricrocin--an ectomycorrhizal siderophore of Cenococcum geophilum.

The ectomycorrhizal fungus Cenococcum geophilum was grown in low-iron medium and the excreted siderophores were extracted, purified and analyzed by HPLC. The principal hydroxamate siderophore produced, was identified as ferricrocin as confirmed by analytical HPLC, FAB-mass spectrometry and 1H- and 13C-NMR spectra. Although the occurrence of ferricrocin has been shown earlier to occur in the ericoid mycorrhizal fungi, this is the first report of ferricrocin in a true ectomycorrhizal fungus which is taxonomically related to the ascomycetes.

Production of the phytotoxic metabolite, ferricrocin, by the fungus Colletotrichum gloeosporioides.

A siderophore was isolated as a non-specific phytotoxic compound from a culture of Colletotrichum gloeosporioides isolated from infected blackberry. This siderophore was identified as ferricrocin by NMR, IR, MS, and CD spectra. The phytotoxic activities of ferricrocin and deferriferricrocin were compared.

Characterization of siderophores from Ustilago maydis.

Two siderophores, ferrichrome and ferrichrome A, were found in cultures of Ustilago maydis (DC) Corda. Both siderophores were found intracellularly and extracellularly. Their authenticity was confirmed by thin layer chromatography, HPLC, UV-visible spectrometry, paper electrophoresis, amino acid analysis, NMR and fast atom bombardment mass spectroscopy. Regulation of siderophore production by iron was examined. Repression of biosynthesis of extracellular siderophores occurred at 10(-5) M iron. Ferrichrome was found intracellularly at all iron concentrations employed; in general, ferrichrome A was not found to be cell-associated.

Separation of ferrichromes and other hydroxamate siderophores of fungal origin by reversed-phase chromatography.

Iron(III) chelates of nineteen trihydroxamate siderophores of fungal origin, including ferrichromes, coprogen and triacetylfusarinine C, were separated on a preparative scale with a reversed-phase column using the octadecyl silica gels LRP-1 or LRP-2 as the stationary phase and a water-methanol gradient as the mobile phase. Using this system in combination with silica gel column chromatography, most siderophores can be obtained in pure form. Factors affecting the mobility of these compounds in the reversed-phase system are discussed.

Malonichrome, a new iron chelate from Fusarium roseum.

The predominant iron chelates, or siderochromes, produced by the fungus, Fusarium roseum during culture periods up to seven days are the ester type fusarinine compounds. During longer periods of incubation, the fusarinine compounds completely disappear from the culture medium and are replaced by a new siderochrome. The new compound has been isolated, purified, and its structure determined. It is a cyclic hexapeptide containing one residue of L-alanine, two residues of glycine and three residues of delta-N-hydroxyornithine. The hydroxylamino groups of the ornithine residues are acylated with 3 mol of malonic acid to form a negatively charged ferrichrome type chelate. The circular dichroism spectrum indicates that the stereochemistry about the iron is lambda-cis. This compounds, which we name malonichrome, is not an efficient iron donor to F. roseum nor does it show growth factor activity towards Arthrobacter flavescens.

[Metabolic products of microorganisms. 150. Ferricrocin, triacetylfusigen and other sideramines from fungi of the genus Aspergillus, group Fumigatus (author's transl)]. / Stoffwechselprodukte von Mikroorganismen 150. Mitteilung. Ferricrocin, Triacetylfusigen und andere Sideramine aus Pilzen der Gattung Aspergillus, Gruppe Fumigatus

The sideramines of 14 species of the genus Aspergillus, group Fumigatus, were isolated and analyzed. Ferricrocin was identified in 11 extracts, ferrichrome in 2 others. A lipophilic sideramine produced by 4 species was identified as triacetylfusigen, another minor component as triacetylfusigen B. Six species produced an alanine-containing sideramine, which may be identical with ferrichrom C.