1887

Abstract

The intestinal spirochaete causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from strain 95-1000 cells by osmotic lysis in dHO followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; =1·18 g cm) and a low-density fraction (LDMV; =1·12 g cm). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa OM protein) and was used as a source of antigens to produce mAbs. Five -specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a 95-1000 genome library with the mAb and was expressed in . Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within .

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2004-04-01
2019-10-24
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