Iron uptake in Ustilago maydis: studies with fluorescent ferrichrome analogues

Abstract

Iron uptake by the phytopathogenic fungus Ustilago maydis was studied using synthetic biomimetic ferrichrome analogues and their fluorescently labelled derivatives as structural and dynamic probes, respectively. The use of structurally distinct analogues enabled determination of the structural requirements for recognition by the fungal iron-uptake system. The application of fluorescently labelled derivatives which convert from a non-fluorescent to a fluorescent state upon iron (III) release enabled monitoring of iron uptake in real time both fluorimetrically and microscopically. Different rates of 55Fe uptake were found for two structurally distinct synthetic analogues, B9 and B5, which differ in their amino acid building blocks. B9 mediated uptake of 55Fe at a higher rate than B5. The behaviour of the fluorescent derivatives B9-Ant (anthracene-labelled B9) and B5-Ant (anthracene-labelled B5) paralleled that of their non-labelled precursors. Exposure of fungal cells to B9-Ant led to a higher increase of fluorescence in the medium than exposure to B5-Ant, indicating a more effective iron uptake from B9-Ant. By using fluorescence microscopy it was possible to trace the label of B9-Ant. Fluorescence was localized in regularly shaped vesicles in the treated cells. The rate of fluorescence appearance within the cells lagged behind the rate of iron uptake, suggesting use of the siderophores for iron storage.

Desferrioxamine-mediated Iron Uptake in Saccharomyces cerevisiae

“…The sequence and predicted structure of the ARN family of transporters, however, do not resemble those of prokaryotic siderophore transporters; presumably eukaryotes have evolved distinct mechanisms for siderophore-mediated iron uptake. The fungus Ustilago maydis exhibits two different pathways of siderophore-mediated iron uptake (32,33). The native siderophore ferrichrome enters the cell as an iron chelate and over time accumulates in vesicles in the iron-free form.…”

Identification of a Candida albicans Ferrichrome Transporter and Its Characterization by Expression inSaccharomyces cerevisiae

“…These results suggest that Ga-Fc competes with Fe-Fc for a recognition site on CaArn1p. Ferrichrome synthetic analogues are recognized and taken up by the Ustilago maydis ferrichrome uptake system (15,16). These analogues were not taken up by pmet-CaARN1-transformed ⌬fet3⌬arn1-4 cells, as ascertained by growth, radiotracer experiments, and fluorescence microscopy (data not shown).…”

“…This competition was transient as removal of Ga-Fc alleviated the block in 59 Fe-Fc uptake. Analogues of ferrichrome were previously shown to be biologically active in the ferrichrome-producing fungus U. maydis (15,16). While it has been suggested that S. cerevisiae can accumulate these siderophores and their fluorescently labeled derivatives (31), we have not seen uptake or growth promotion by the fluorescent ferrichrome analogues in cells expressing either the CaARN1p or the endogenous S. cerevisiae ARN1p.…”

“…Siderophore complexes were prepared by overnight incubation of desferri-siderophores with FeCl 3 or Ga (NO 3 ) 3 at a ratio of 1.0:0.9. All siderophore complexes were filter-sterilized (0.2 M filter) (15,16).…”

Compostos de ferro de interesse farmacológico: avaliação da estabilidade, toxicidade em organismos aquáticos, transporte em células e capacidade de gerar reservatórios de ferro lábil

“…Outros sideróforos naturais ou sintéticos, diferentes do DFO, podem ser acoplados a sondas fluorescentes ou tornados intrinsicamente fluorescentes na busca de sintetizar novas sondas fluorescentes específicos de ferro 83,[86][87][88][89][90] …”