The iron-siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11-Pro16 beta-hairpin region in the recognition mechanism.
The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.
Destoumieux-Garzón
Delphine
D
Chimie et Biochimie des Substances Naturelles, CNRS UMR 5154, Muséum National d'Histoire Naturelle USM 502, Département Régulations Développement et Diversité Moléculaire, 63 rue Buffon, 75005 Paris, France. ddestoum@mnhn.fr
Duquesne
Sophie
S
Peduzzi
Jean
J
Goulard
Christophe
C
Desmadril
Michel
M
Letellier
Lucienne
L
Rebuffat
Sylvie
S
Boulanger
Pascale
P
D020816:Amino Acid Motifs; D001419:Bacteria / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D001430:Bacteriocins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D008958:Models, Molecular; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism; D011991:Receptors, Virus / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D017906:Siphoviridae
D001425:Bacterial Outer Membrane Proteins; D001430:Bacteriocins; D029968:Escherichia coli Proteins; C050900:FhuA protein, E coli; D011956:Receptors, Cell Surface; D011991:Receptors, Virus; C020411:microcin