The Treponema denticola major sheath protein is predominantly periplasmic and has only limited surface exposure

The recent discovery that the Treponema pallidum genome encodes 12 ortholog s of the Treponema denticola major sheath protein (Msp) prompted us to reex amine the cellular location and topology of the T, denticola polypeptide. E xperiments initially were conducted to ascertain whether Msp forms an array on or within the T. denticola outer membrane. Transmission electron micros copy (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 prote ins but no array. In contrast, a lattice-like structure was observed in ves icles released from mildly sonicated treponemes; combined EM and biochemica l 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-sectio ned, intact treponemes also demonstrated that the preponderance of antigen was unassociated with the outer membrane. Lastly, immunofluorescence analys is of treponemes embedded in agarose gel microdroplets revealed that only m inor portions of Msp are surface exposed. Taken as a whole, our findings ch allenge the widely held belief that Msp forms an array within the T. dentic ola outer membrane and demonstrate, instead, that it is predominantly perip lasmic with only limited surface exposure. These findings also have implica tions for our evolving understanding of the contribution(s) of Msp/Tpr orth ologs to treponemal physiology and disease pathogenesis.