TheTreponema denticolaMajor Sheath Protein Is Predominantly Periplasmic and Has Only Limited Surface Exposure

Abstract

The recent discovery that the Treponema pallidum genome encodes 12 orthologs of the Treponema denticola major sheath protein (Msp) prompted us to reexamine the cellular location and topology of the T. denticola polypeptide. Experiments initially were conducted to ascertain whether Msp forms an array on or within the T. denticola outer membrane. Transmission electron microscopy (EM) of negatively stained and ultrathin-sectioned organisms failed to identify a typical surface layer, whereas freeze-fracture EM revealed that the T. denticola outer membrane contains heterogeneous transmembrane proteins but no array. In contrast, a lattice-like structure was observed in vesicles released from mildly sonicated treponemes; combined EM and biochemical analyses demonstrated that this structure was the peptidoglycan sacculus. Immunoelectron microscopy (IEM) subsequently was performed to localize Msp in T. denticola. Examination of negatively stained whole mounts identified substantial amounts of Msp in sonicated organisms. IEM of ultrathin-sectioned, intact treponemes also demonstrated that the preponderance of antigen was unassociated with the outer membrane. Lastly, immunofluorescence analysis of treponemes embedded in agarose gel microdroplets revealed that only minor portions of Msp are surface exposed. Taken as a whole, our findings challenge the widely held belief that Msp forms an array within the T. denticola outer membrane and demonstrate, instead, that it is predominantly periplasmic with only limited surface exposure. These findings also have implications for our evolving understanding of the contribution(s) of Msp/Tpr orthologs to treponemal physiology and disease pathogenesis.

Cloning and characterization of a major surface protein (MspTL) of Treponema lecithinolyticum associated with rapidly progressive periodontitis

“…SDS^PAGE analysis and immunoblotting revealed MspTL as the major component of the OMF released from the whole cells by gentle vortexing. It is well known that treponemal outer membranes are fragile and washing them in PBS releases outer membranes [2].…”

Outer sheath associated proteins of the oral spirochete Treponema maltophilum

“…The T. denticola Msp protein was shown to be an outer membrane porin, with binding properties to host cell surface proteins [4,5,12]. Further it was reported that the Msp protein forms a hexagonal array in the outer sheath [4], however, this could not be con¢rmed by a recent publication [2]. Electron microscopic analysis of both, complete bacteria as well as isolated outer membranes of T. maltophilum never revealed the existence of a hexagonal array (data not shown).…”

Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola

“…Rosen et al [24] observed that the vesicles released from T. denticola were constituted of components similar to those of the outer sheath and they included dentilisin. On the other hand, Caimano et al [10] frequently observed vesicles which did not react with anti-Msp. To evaluate the mechanism of the virulence of the surface protein, further analysis is required to determine whether the surface component is released by vesicles from live cells or by breakdown of the cells.…”

“…Msp has been believed to be located in the outer sheath, to be strongly exposed at the surface and to be visible as a hexagonal array structure in the outer sheath by electron-microscopy [14,15]. Recently, Caimano et al [10] demonstrated new electron-microscopic ¢ndings about the location of Msp and the array structure. An array structure is typically observed in bacteria which have an S-layer.…”

“…Many attempts to evaluate such pathogenic factors in T. denticola as adherence factors, immunomodulating factors and cytopathic factors have been reported [6^9]. Recently, the surface structures of T. denticola have been characterized [10,11], and genetic analysis of such surface components as the major outer sheath protein (Msp; [12]) and prolyl-phenylalanine speci¢c protease (dentilisin) [13] has helped to elucidate their structure and role in pathogenicity. These reports emphasize the importance of the bacterial surface to the pathogenic e¡ects.…”