Neutrophil activation by nascent FimH subunits of type 1 fimbriae purified from the periplasm of Escherichia coli.
Previous studies of type 1 fimbriae of Escherichia coli have implicated FimH, a minor subunit, as the determinant of its mannose binding property. Structure-function analysis of FimH has not been possible because of the difficulty in obtaining adequate amounts of the subunit from type 1 fimbriae. We have obtained nascent FimH that has not been incorporated into the fimbrial structure from the periplasm of an E. coli strain expressing the cloned fimH gene. Nascently translocated FimH was initially degraded in the periplasm; however, when co-expressed with FimC, a putative fimbrial chaperone, the FimH molecules were stabilized and readily isolated from the periplasmic extract by fractionation on a sodium dodecyl sulfate-polyacrylamide gel followed by electroelution of the FimH band from the gel. The eluted protein was purified to homogeneity by affinity chromatography on a mannose-Sepharose column. Purified FimH displayed the same mannose-inhibitable binding to human neutrophils as type 1 fimbriated bacteria, including triggering an oxidative burst with concomitant release of reactive oxygen metabolites. In addition, inert microspheres coated with FimH, but not those coated with bovine serum albumin, were phagocytosed by neutrophils. These data provide direct evidence that FimH is the determinant on type 1 fimbriae which is responsible for mediating mannose-specific adherence and that isolated FimH is a potent activator of human neutrophils.
Tewari
R
R
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.
MacGregor
J I
JI
Ikeda
T
T
Little
J R
JR
Hultgren
S J
SJ
Abraham
S N
SN
D018830:Adhesins, Escherichia coli; D000942:Antigens, Bacterial / Q000276:immunology*; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004705:Endocytosis; D004926:Escherichia coli / Q000276:immunology*; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000276:immunology*; D008854:Microscopy, Electron; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D011498:Protein Precursors / Q000276:immunology; D016897:Respiratory Burst
D018830:Adhesins, Escherichia coli; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011498:Protein Precursors; C058769:fimH protein, E coli; D036781:Fimbriae Proteins