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Abstract
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.
Highlights
From the Departments of $Molecular Microbiologyand 11Pathology, Washington University School of Medicine and the Departments of §Pathologyand WMedicine, Jewish Hospital, St
Evidence implinascent FimH that has not been incorporated into the fimbrial structure from the periplasm of an E. coli strainexpressingthe cloned fimH gene.Nascently translocated FimH was initially degraded in the periplasm; when co-expressed with FimC,a putative fimbrial chaperone, the FimH molecules were cating FimH as the mannose-binding moiety on type 1 fimbriae is indirect: (i) from geneticstudies showing that mutants lacking fimH,but notgenes encodingother fimbrial subunits, fail to bind toeucaryotic cells [11,12,13], and (ii) from observationsthatantibodiesdirectedatFimH,butnotatother stabilized and readily isolated from the periplasmic fimbrial subunits, block cellular binding of type 1 fimbriated extract by fractionation on a sodium dodecyl sulfate- bacteria [3, 6]
Interaction of bacteria with these spheres coated with FimH, but not those coated with cellscould determinenot only bacterial survival butthe bovine serum albumin, were phagocytosed by neutrophils
Summary
Suspension (lo6cells/ml) into each chamber of an eight-chamber slide (Nunc Inc.) which was placed in a humid incubator at 37 "C for 30. They were placed on each monolayer and incubated for 1 h at fragment isolated from E. coli J96 which encodes all of the genes 37 "C on a horizontal orbital shaker(set at 60 rpm)ina moist necessary for the expression of functional type 1 fimbriae [26]. After incubation, unattached bacteria wereremoved by Plasmid pUT2002 was obtained by deleting a 1.2-kb fragment con- gently rinsing the monolayers in cold Hanks' balanced salt solution. By ligating the 1.2-kb PuuII fragment containing fimH intomultiple Adherent bacteria were enumerated under bright-field microscopy, cloning site of pUC plasmid. Cloning a 1.3-kb BamHI-EcoRI fragment containing fimC from the Electron Microscopy-A mixture of neutrophils with adhering mitype 1 gene cluster into kanamycin-resistant pMMB91 which is the crospheres prepared as described above was washed and fixed in 2%.
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