Isolation and characterization of Bacteroides nodosus fimbriae: structural subunit and basal protein antigens

Abstract

We examined the isolation of fimbriae from Bacteroides nodosus. It was found that the best preparations were obtained from the supernatant of washed cells cultured on solid medium, from which fimbriae could be recovered in high yield and purity by a simple one-step procedure. Analysis of such preparations by sodium dodecyl sulfate gel electrophoresis showed that greater than 98% of the protein consisted of fimbrial structural subunits whose molecular weight was ca. 17,000. These preparations also usually exhibited minor contamination with a polypeptide of ca. 80,000 molecular weight, as well as trace amounts of lipopolysaccharide. Attempts to release additional fimbriae by the traditional means of subjecting the bacterial cells to physical stress, such as shearing or heating, resulted primarily in an increase in the level of contamination, without significant gain in the yield of fimbriae. Removal of the 80,000-dalton component could not be achieved by any of a variety of techniques normally used in fimbriae purification, including isoelectric precipitation, MgCl2 precipitation, and CsCl gradient ultracentrifugation, implying a direct physical association with the fimbrial strand. Electron micrographs of fractions containing this protein show cap-shaped structures attached to the ends of what appeared to be fimbrial stubs. These observations suggest that the 80,000-dalton polypeptide may actually constitute the basal attachment site which anchors the fimbria to the outer membrane, analogous to a similar protein recently described in enterotoxigenic strains of Escherichia coli. In B. nodosus, this 80,000-* Corresponding author. which is then followed by a lengthy series of purification steps (e.g., see reference 10). However, using these procedures, we noted substantial contaminants and, in particular, the presence and persistence of a protein of about 80,000 molecular weight. Our results indicate, first, that the current methods used for the production offimbriae from B. nodosus may be inappropriate, even counterproductive, and we present a simplified protocol for the isolation of fimbriae in high purity. Second, our evidence suggests that the 80,000dalton component may actually be physically associated with fimbriae in vivo and in vitro, and that it probably represents the basal protein which anchors the fimbrial strand to the outer surface of the cell.MATERIALS AND METHODS Bacterial culture. Bacteroides nodosus 198, originally obtained from the Commonwealth Scientific and Industrial Research Organization McMaster Laboratory (Sydney, Australia) culture collection, was used in these studies. This strain is the prototype assigned to serogroup A, subtype 1 and has been reclassified as VCS 1001 (3). Strain VCS 1006 (serogroup B, subtype 1) was also used in some experiments. Seeding cultures were purified from freeze-dried samples by two subcultures on 4% hoof-agar (HA) medium (35). For large-scale cultures, cells were grown on 2% HA medium and harvested by being scraped into 10 mM sodium phosphate buffer (pH 7.2) co...

Cloning and expression in Escherichia coli of the gene encoding the structural subunit of Bacteroides nodosus fimbriae

“…nodosus fimbrial strands consist of a single repeated polypeptide subunit, which in serotype Al has a molecular * Corresponding author. weight of about 17,500 (10,17,19); in other serotypes the size appears to vary (15,17). Isolated fimbrial preparations also contain another significant component, a polypeptide of ca.…”

Complementation Analysis of the Dichelobacter nodosus fimN , fimO , and fimP Genes in Pseudomonas aeruginosa and Transcriptional Analysis of the fimNOP Gene Region

“…Differential expression of fim genes in D. nodosus. D. nodosus cells produce fewer fimbriae when they are grown in liquid media than when they are grown on solid agar surfaces (32). To determine whether this effect was due to differential transcription of the fimbrial subunit gene, fimA, or the fim operon, dot blots were performed on RNA extracted from D. nodosus cells grown for ϳ84 h in Eugonbroth or on Eugon agar, each of which contained 10% horse serum.…”

A multiple site‐specific DNA‐inversion model for the control of Ompi phase and antigenic variation in Dichelobacter nodosus

“…Outer membrane complex (OMC) was prepared by the method of Heckels (1977). Fimbriae for Western blot analysis were isolated as described by Mattick et aL (1984). For isolation of the c. 75kDa polypeptide that co-purifies with the fimbriae, the fimbriae were further purified by banding in CsCI, as described by Every (1979).…”