Lactoferrin-binding proteins in Shigella flexneri.
The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri.
Tigyi
Z
Z
Department of Medical Microbiology, Malmö General Hospital, Sweden.
Kishore
A R
AR
Maeland
J A
JA
Forsgren
A
A
Naidu
A S
AS
D001422:Bacterial Adhesion / Q000187:drug effects; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature / Q000009:adverse effects; D015151:Immunoblotting; D007781:Lactoferrin / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000032:analysis; D018272:Porins; D012763:Shigella flexneri / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D008070:Lipopolysaccharides; D018272:Porins; D013861:Thiocyanates; D012965:Sodium Chloride; D014508:Urea; D007781:Lactoferrin; C009941:potassium thiocyanate