Importance of the disulfide bridges in the antibacterial activity of human hepcidin.
Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25, and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity.
Univ. Bordeaux, BPRVS, F-33000 Bordeaux, France.
Agnès
A
Hocquellet
Caroline
C
le Senechal
Bertrand
B
Garbay
D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000187:drug effects; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D064451:Hepcidins; D006801:Humans; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D004220:Disulfides; C578702:HAMP protein, human; D064451:Hepcidins