Journal of Molecular Biology
Volume 403, Issue 4, 5 November 2010, Pages 546-561
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Cellular Architecture of Treponema pallidum: Novel Flagellum, Periplasmic Cone, and Cell Envelope as Revealed by Cryo Electron Tomography

https://doi.org/10.1016/j.jmb.2010.09.020Get rights and content

Abstract

High-resolution cryo electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member of the spirochetal family. High-resolution cryo-ET reconstructions provided detailed structures of the cell envelope, which is significantly different from that of Gram-negative bacteria. The 4-nm lipid bilayer of both outer membrane and cytoplasmic membrane resolved in 3D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High-resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located cone-shaped structure at both ends of the bacterium. Furthermore, 3D subvolume averages of periplasmic flagellar motors and flagellar filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Our findings provide the most detailed structural understanding of periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and to escape host immune responses.

Introduction

Treponema pallidum subsp. pallidum is the causative agent of syphilis, a sexually transmitted disease with more than 12 million new cases worldwide each year.1 Since 2000, the reported cases of primary syphilis and secondary syphilis have been rising annually in the United States.2 Of major concern is the recognition that syphilis infection greatly increases susceptibility to human immunodeficiency virus infection.3 In addition, closely related organisms cause endemic syphilis (T. pallidum subsp. endemicum), yaws (T. pallidum subsp. pertenue), pinta (Treponema carateum), and venereal spirochetosis in rabbits (Treponema paraluiscuniculi).4 The genome sequences of several of these organisms have been determined.5, 6 T. pallidum continues to be an enigmatic pathogen because of the lack of readily identifiable virulence determinants and the poorly understood pathogenesis of the disease. This deficiency is due primarily to the inability to culture members of this group of obligate pathogens continuously in vitro.7

T. pallidum is a member of the Spirochaetaceae, a biomedically important bacterial phylum that includes the etiological agents of Lyme disease (Borrelia burgdorferi)8 and leptospirosis (Leptospira interrogans).9 A striking feature of T. pallidum and other spirochetes is their capacity to swim efficiently in a highly viscous gel-like environment, such as connective tissues, where most externally flagellated bacteria are slowed or stopped.10 Therefore, motility is likely to play an important role in the widespread dissemination of spirochetal infections and in the establishment of chronic disease.10, 11 Motility-associated genes are shared by both T. pallidum and B. burgdorferi, and an in-depth comparative analysis of these spirochetes may greatly enhance our understanding of the fundamental physiology of these important pathogens.5, 12

T. pallidum has a spiral shape with a length ranging from 6 μm to  15 μm and a diameter of ∼  0.2 μm.11, 13, 14, 15, 16 The protoplasmic cylinder is surrounded by a cytoplasmic membrane, which is enclosed by a loosely associated outer membrane. A thin layer of peptidoglycan between the membranes provides structural stability while permitting flexibility. The flagella are located in the periplasmic space, between the cytoplasmic membrane and the outer membrane. Bundles of flagella originate from flagellar motors at both ends of the organism, wind around the flexible protoplasmic cell cylinder, and overlap in the middle. T. pallidum contains multiple chemotaxis genes encoding methyl-accepting chemotaxis proteins.5 Treponema species also contains cytoplasmic filaments arranged in a ribbon configuration that spans the entire length of the cell.17, 18, 19, 20 cfpA, the gene encoding the ∼ 79-kDa major subunit of the cytoplasmic filaments, is highly conserved in Treponema species. In Treponema denticola or Treponema phagedenis, mutation of cfpA results in defective cell division in which two or more cytoplasmic cylinders are encased in a single outer membrane.17 There are at least 46 putative lipoproteins in T. pallidum;21 other than the 47-kDa carboxypeptidase and multiple ABC transporter periplasmic binding proteins, the functions of these lipoproteins are largely unknown.11 T. pallidum also has a paucity of integral membrane proteins in its outer membrane and has been referred to as the “stealth pathogen” because of the scarcity of antigens on its outer surface.2, 22, 23

Flagella play an essential role in the motility, morphology, and biology of spirochetes and other organisms.10 Flagellar structure and function have been extensively studied using Escherichia coli and Salmonella enterica as model systems. Flagellar motion is powered by a proton-driven or sodium-ion-driven rotary motor embedded in the cytoplasmic membrane, but the precise mechanisms of flagellar motor rotation and reversal remain to be determined.24, 25, 26, 27, 28 The periplasmic flagellar assemblies of T. pallidum are expected to generally resemble those of external flagella, owing to the high degree of similarity between flagellar gene homologs. In recent years, there has been progress in understanding the periplasmic-flagella-driven motility of B. burgdorferi and other spirochetes.29 The motility of B. burgdorferi results from the coordinated asymmetric rotation of the flagella at both ends of the cell.30 The T. pallidum flagellar filaments are composed of several major proteins, including flagellar core proteins FlaB1, FlaB2, and FlaB3, and the sheath protein FlaA.31, 32, 33 However, the structural details of periplasmic flagella remain unknown.

The cellular architectures of several spirochetes (including T. pallidum, Treponema primitia, T. denticola, and B. burgdorferi) have been studied extensively using cryo electron tomography (cryo-ET) analysis.34, 35, 36, 37 Cryo-ET has emerged as a three-dimensional (3D) imaging technique used to visualize cellular and subcellular structures at the molecular level.38, 39 The distinct advantage of cryo-ET is its potential to elucidate the structure of cellular components in situ without fixation, dehydration, embedding, or sectioning artifacts. The power of cryo-ET has been significantly enhanced by 3D subvolume averaging and classification techniques.40, 41, 42, 43 We recently used high-throughput cryo-ET of B. burgdorferi to determine the molecular architecture of intact flagellar motor at  a resolution of 3.5 nm.44

In this study, we investigated infectious T. pallidum organisms and their periplasmic flagella in situ by utilizing high-throughput cryo-ET. Cryo-ET reconstructions from 304 frozen–hydrated organisms generally support the recently published results of Izard et al. but provide a significant amount of novel structural information regarding the detailed structures of the cell envelope, the localization of chemoreceptor arrays, and the molecular architecture of flagellar filaments and flagellar motors in situ.35 Abundant lipoproteins studding the outer leaflet of the cytoplasmic membrane were visualized for the first time; the proteinaceous nature of this layer was verified by its dissolution following proteinase K treatment of partially disrupted organisms. A periplasmically located cone-shaped structure was observed at the ends of each organism; this unique feature appears to be composed of lipoproteins arranged in a helical lattice adjacent to the outer membrane. Furthermore, a comparative analysis of B. burgdorferi and T. pallidum provides insight into the common cellular structures of spirochetes and also exhibits some unique pathogenesis-related features of these two pathogens.

Section snippets

Cryo-ET of intact T. pallidum cells

T. pallidum cells (n = 304) were imaged under optimal conditions to obtain high-resolution structures using high-throughput cryo-ET. The organism typically tapers from an approximate diameter of 0.2 μ m to 0.1 μ m near the end of the cell, as shown in a representative image (Fig. 1a). Similar to B. burgdorferi and other spirochetes examined previously, the image from a T. pallidum cell (Fig. 1a) illustrates the outer membrane, the cytoplasmic membrane, and a thin peptidoglycan layer, with the

Discussion

The pathogenesis of T. pallidum is poorly understood at the molecular level, partly due to the inability to culture the organism continuously in vitro. Innovative approaches are consequently needed to study the fundamental biology of this enigmatic spirochete. Cryo-ET is well suited for the elucidation of the 3D cellular architecture of intact organisms at the molecular level. We are able to achieve high-resolution 3D reconstructions of intact organisms under optimal imaging conditions, such as

Ethics statement

All procedures involving rabbits were reviewed and approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston.

Cryo-ET of intact T. pallidum

T. pallidum subsp. pallidum Nichols was extracted from rabbit testes after intratesticular infection; in some experiments, the organisms were further purified by Percoll density gradient centrifugation, as described previously.20 Freshly prepared motile T. pallidum was centrifuged and resuspended in 20 μ l of phosphate-buffered saline (PBS) at a

Acknowledgements

We thank Drs. James K. Stoops, Angel Paredes, Hanspeter Winkler, Ken Taylor, and Douglas Botkin for their comments and suggestions. We also thank Matthew Swulius for advice and assistance with immunogold labeling. This work was supported, in part, by Welch Foundation grant AU-1714 and National Institutes of Health grant 1R01AI087946 (to J.L.). Maintenance of the Polara electron microscope facility was supported by the Structural Biology Center at the University of Texas Medical School at

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