| Abstract: | Cryo-electron tomography (CET) was used to examine the native cellular organization of Treponema pallidum, the syphilis spirochete. T. pallidum cells appeared to form flat waves, did not contain an outer coat and, except for bulges over the basal bodies and widening in the vicinity of flagellar filaments, displayed a uniform periplasmic space. Although the outer membrane (OM) generally was smooth in contour, OM extrusions and blebs frequently were observed, highlighting the structure''s fluidity and lack of attachment to underlying periplasmic constituents. Cytoplasmic filaments converged from their attachment points opposite the basal bodies to form arrays that ran roughly parallel to the flagellar filaments along the inner surface of the cytoplasmic membrane (CM). Motile treponemes stably attached to rabbit epithelial cells predominantly via their tips. CET revealed that T. pallidum cell ends have a complex morphology and assume at least four distinct morphotypes. Images of dividing treponemes and organisms shedding cell envelope-derived blebs provided evidence for the spirochete''s complex membrane biology. In the regions without flagellar filaments, peptidoglycan (PG) was visualized as a thin layer that divided the periplasmic space into zones of higher and lower electron densities adjacent to the CM and OM, respectively. Flagellar filaments were observed overlying the PG layer, while image modeling placed the PG-basal body contact site in the vicinity of the stator-P-collar junction. Bioinformatics and homology modeling indicated that the MotB proteins of T. pallidum, Treponema denticola, and Borrelia burgdorferi have membrane topologies and PG binding sites highly similar to those of their well-characterized Escherichia coli and Helicobacter pylori orthologs. Collectively, our results help to clarify fundamental differences in cell envelope ultrastructure between spirochetes and gram-negative bacteria. They also confirm that PG stabilizes the flagellar motor and enable us to propose that in most spirochetes motility results from rotation of the flagellar filaments against the PG.Spirochetes are an ancient and extremely successful eubacterial phylum characterized by distinctive helical or planar wave-form morphology and flagellar filaments confined to the periplasmic space (55, 87). Spirochetes from the genera Leptospira, Treponema, and Borrelia are highly invasive pathogens that pose public health problems of global dimensions (1, 6, 57, 109). Treponema denticola and numerous other treponemal species, most of which remain uncultivated, are major components of the polymicrobial biofilms that cause periodontal disease (34, 56) and also have been implicated as risk factors for atherosclerosis (4, 125). The treponemal symbionts that dwell in the hindguts of termites, where they provide their insect host with essential nutrients (10), are one of the most striking examples of the extraordinary biodiversity achieved by spirochetes. It is readily apparent, therefore, that in the course of their complex evolution, spirochetes have exploited a basic ultrastructural plan to accommodate an immense spectrum of metabolic activities and lifestyles, both commensal and pathogenic.Venereal syphilis is a multistage, sexually transmitted disease caused by the noncultivatable spirochete Treponema pallidum. Following inoculation, usually in the genital region, T. pallidum disseminates via lymphatics and blood to diverse organs, where it can establish persistent, even life-long, infection (68, 97). Over the years there has been great interest in defining ultrastructural features of the syphilis spirochete that might contribute to syphilis pathogenesis (58, 64, 84, 120, 121). Classic electron microscopy studies established that T. pallidum possesses a characteristic spirochete ultrastructure consisting of outer and cytoplasmic membranes and periplasmic flagellar filaments originating from cytoplasmic membrane-associated, subterminal basal bodies (55, 58). Hovind-Hougen (58) identified a putative peptidoglycan (PG) layer surrounding the cytoplasmic membrane (CM), and she noted that the end of the bacterium contains a distinct structural entity which she speculated mediates polar attachment to mammalian cells and extracellular matrix components. Freeze-fracture analysis has shown that the T. pallidum outer membrane (OM) contains a lower density of membrane-spanning proteins than its counterparts in either gram-negative bacteria or cultivatable spirochetes (99, 118), and it is thought that the paucity of surface-exposed antigenic targets resulting from this unusual OM ultrastructure is an important element of the spirochete''s strategy for immune evasion (14, 93, 97).In the more than 10 years since the publication of the T. pallidum genomic sequence made available a much-needed parts list for the bacterium (44), we have learned comparatively little about how these components are organized to create this extremely virulent and immunoevasive pathogen. Cryo-electron tomography (CET) has emerged as a powerful methodology for bridging the gap between protein-protein interactions and cellular architecture (70, 71). With this technique, thin films of cells are vitreously frozen to preserve cell structure in a close-to-native state, thereby avoiding chemical fixation, dehydration, and staining artifacts typically associated with conventional electron microscopy (EM). A series of images acquired as the sample is progressively tilted in an electron microscope are used to generate a three-dimensional (3D) reconstruction of the intact cell. In recent years, investigators have used CET to examine a variety of eukaryotic and prokaryotic cell types (70, 73, 77). With respect to spirochetes, CET has been used to visualize the intact flagellar motors of Treponema primitia (79) and Borrelia burgdorferi (67, 72); novel internal and external structural features of T. denticola (60); Treponema primitia (80), B. burgdorferi (66), and Leptospira interrogans (74); the flat ribbon configuration of B. burgdorferi periplasmic flagella (18); and the defects created in B. burgdorferi OMs when organisms are incubated with a borreliacidal monoclonal antibody (69). In the present study, we used CET to examine the native cellular organization of T. pallidum. These analyses demonstrated, not surprisingly, that T. pallidum shares many structural features with T. denticola while, at the same time, calling attention to the fluidity and dynamism of the syphilis spirochete''s cell envelope. Our study also revealed that T. pallidum cell ends possess an unexpected degree of structural complexity and diversity compared to those of other spirochetes examined to date by CET. Lastly, our work has clarified the location of the PG layer within the periplasmic space and its spatial relationship to the motility apparatus, which are prerequisites for understanding spirochete movement and, by extension, invasiveness. As a whole, the information obtained underscores and clarifies fundamental differences in cell envelope composition and organization between T. pallidum, as well as other pathogenic spirochetes, and the model gram-negative bacterium, Escherichia coli. |
