Isolation and Characterization of Cytoplasmic Fibrils from Treponemes

Abstract

Electron microscopy of Triton X-100-treated whole cells of an oral treponeme, Treponema sp. strain E-21, revealed that six cytoplasmic fibrils (CFs) helically wound as a bundle in the cytoplasm. The CFs were isolated and purified by disruption and solubilization of the cells followed by CsCl density gradient centrifugation. The purified CF preparation contained mostly fibrils of about 9 nm in width and very small amounts of thinner strands of about 3 nm in diameter. The CFs were apparently seen to be a tubular structure, but the isolated CFs had narrowed sites of about 4-5 nm in width lacking lumen-like images, possibly representing twisted sites. Thus, the CF did not seem to be a tubular structure. The purified CFs were composed of one major 82 kDa protein and a few minor proteins. The CFs were destructed by treatment with proteases, 8 M urea or 4 M guanidine hydrochloride. Very low tyrosine content (0.76 mol %) and lack of methionine were characteristic features for the 82 kDa protein. The CF preparations from the other five treponemes including Treponema phagedenis and T. denticola also had 82 kDa proteins as a major component, and the 82 kDa proteins of all of the treponemes had a common antigen when examined by using antiserum against the 82 kDa protein from Treponema sp. strain E-21. Furthermore, the 82 kDa protein was demonstrated to be a principal component of the CFs of all the treponemes by immunoelectron microscopy.

Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum

“…2), similar to cytoplasmic filament subunit proteins described by Masuda and Kawata (25) for T. phagedenis and several other spirochetes. The single-and two-dimensional gel migration characteristics of this protein are very similar to those of the T. pallidum polypeptide previously described as TpN83, which comprises 3 to 5% of the total cellular protein of T. pallidum (29,32).…”

“…Little is known about the mechanism of assembly or the possible arrangement of subunits within the cytoplasmic filaments. Filaments from nonpathogenic treponemes are stable in the presence of 2% Triton X-100, 2% sodium deoxycholate, and 2 M urea but dissociate in the presence of 1.3% SDS, 6 M urea, 4 M guanidine hydrochloride, or highly acidic or basic conditions (14,25). Thus, noncovalent forces are responsible for the integrity of the filament structure.…”

“…(ii) Their location strongly implies a role of cytoplasmic filaments in the motility of the cell. However, Masuda and Kawata (25) previously purified the cytoplasmic filaments from three Treponema strains that were isolated from the oral cavities of patients with pyrrhoea (strains not identified to species level), as well as from T. phagedenis and T. denticola. The procedure they employed included sonic disruption of the cells, treatment of the lysate with Triton X-100, lysozyme, and N-acetylmuramidase to solubilize membranes and peptidoglycan, and (in some separations) CsCl density gradient sedimentation.…”

“…T. pallidum cells (ϳ10 (25). Note the presence of cosedimenting flagellar basal bodies (BB) and basal plates (BP).…”

“…The cytoplasmic filaments of T. phagedenis were purified by the procedure of Masuda and Kawata (25). The major 80-kDa constituent of the T. phagedenis cytoplasmic filaments and the corresponding 80-kDa band in T. pallidum were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, Mass.…”