Submitted to: Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/15/2003
Publication Date: 4/1/2004
Citation: Trott, D.J., Alt, D.P., Zuerner, R.L., Bulach, D.M., Wannemuehler, M.J., Stasko, J.A., Townsend, K.M., Stanton, T.B. 2004. Identification and cloning of the gene encoding BmpC: an outer membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles. Microbiology. 150 (Pt 4):1041-1053. Interpretive Summary: Intestinal bacteria known as spirochetes, in the genus Brachyspira, can cause serious diseases in livestock. Little is known about the bacterium B. pilosicoli that causes intestinal spirochetosis in swine and avian species. The goal of these experiments was to identify proteins located on the surface of this bacterium and that are useful targets for vaccine development and for developing diagnostic tests. A biochemical procedure was devised to isolate proteins from B. pilosicoli cell surfaces. Antibody reagents to detect specific proteins were prepared and shown to react with surface proteins. The gene for one abundant surface protein was cloned and analyzed. These experiments provide a foundation for identifying virulence associated surface proteins of this important pathogen and have generated immunological reagents that will allow us to characterize these bacteria during disease.
Technical Abstract: The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the Brachyspira pilosicoli outer membrane (OM). In order to identify surface exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in distilled water followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into high- and low-density fractions (HDMV and LDMV, respectively). LDMV contained predominantly OM markers and was used as a source of antigens to produce monoclonal antibodies (MAbs). Five B. pilosicoli-specific MAbs reacting with proteins with molecular masses of 23 kDa, 24 kDa, 35 kDa, 61 kDa, and 79 kDa were characterized. The MAbs were used to localize the 23, 24, and 35 kDa proteins to the OM. Detergent phase partitioning in Triton X-114 suggests the 24 kDa and 35 kDa proteins are integral membrane proteins. The gene encoding the abundant, surface exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the MAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein designated BmpC. Evidence was obtained that suggests either the surface-exposed epitope recognized by the Mab on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.