Porins OmpC and PhoE of Escherichia coli as specific cell-surface targets of human lactoferrin. Binding characteristics and biological effects.
The binding of lactoferrin, an iron-binding glycoprotein found in secretions and leukocytes, to the outer membrane of Gram-negative bacteria is a prerequisite to exert its bactericidal activity. It was proposed that porins, in addition to lipopolysaccharides, are responsible for this binding. We studied the interactions of human lactoferrin with the three major porins of Escherichia coli OmpC, OmpF, and PhoE. Binding experiments were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds to the purified native OmpC or PhoE trimer with molar ratios of 1.9 +/- 0.4 and 1.8 +/- 0.3 and Kd values of 39 +/- 18 and 103 +/- 15 nM, respectively, but not to OmpF. Furthermore, preferential binding of lactoferrin was observed on strains that express either OmpC or PhoE. It was also demonstrated that residues 1-5, 28-34, and 39-42 of lactoferrin interact with porins. Based on sequence comparisons, the involvement of lactoferrin amino acid residues and porin loops in the interactions is discussed. The relationships between binding and antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological studies revealed that lactoferrin can act as a blocking agent for OmpC but not for PhoE or OmpF. However, a total inhibition of the growth was only observed for the PhoE-expressing strain (minimal inhibitory concentration of lactoferrin was 2.4 mg/ml). These data support the proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the stability and/or the permeability of the bacterial outer membrane.
Sallmann
F R
FR
Laboratoire de Chimie Biologique et Unité Mixte de Recherche 8576 du CNRS, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France.
Baveye-Descamps
S
S
Pattus
F
F
Salmon
V
V
Branza
N
N
Spik
G
G
Legrand
D
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006801:Humans; D007781:Lactoferrin / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010539:Permeability; D018272:Porins / Q000378:metabolism*; D011485:Protein Binding; D013499:Surface Properties
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; C113724:OmpC protein; C109237:OmpF protein; D018272:Porins; C082922:PhoE protein, E coli; D007781:Lactoferrin