Characterizing the S‐layer structure and anti‐S‐layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X‐ray scattering

Abstract

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22-nm-thick, 2D crystalline surface (S-) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium-host cross-talk. The S-layer is composed of two intercalating glycoproteins (TfsA-GP, TfsB-GP) that are aligned into a periodic lattice. To characterize this unique S-layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and single-molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild-type and S-layer-deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high-resolution imaging of the outer surface of T. forsythia wild-type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti-TfsA antibody coupled to the AFM tip and the S-layer as present on T. forsythia wild-type and mutant cells, displaying TfsA-GP alone. Unbinding forces between the antibody and wild-type cells were greater than with mutant cells. This indicated that the TfsA-GP is not so strongly attached to the mutant cell surface when the co-assembling TfsB-GP is missing. Altogether, the data gained from

Tannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors

“…The ratio of TfsA:TfsB on the cell surface as estimated by spectral counting was very close to 1:1 with 71 ± 11 and 70 ± 10 being identified for TfsA and TfsB respectively (Table S-3.3). The calculated 1:1 ratio is consistent with analyses of the S-layer using AFM and TEM techniques 16,17 . However, the stoichiometry of these proteins in the OMVs was significantly different.…”

Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence

“…1C,D). These findings are supported by non-invasive AFM in vitro imaging of OMV-secreting T. forsythia cells, where cells neither received any chemical treatment nor were subjected to a drying process, which may trigger undesirable biological side effects (Oh et al , 2013). There, OMVs could be clearly visualized in the immediate surroundings as well as lying on top of the imaged T. forsythia cell (Fig.…”

“…Transmission electron microscopy (TEM) of negatively stained and ultrathin-sectioned preparations of T. forsythia cells as well as of isolated T. forsythia OMVs was performed on a Tecnai G2 20 Twin microscope (FEI, Eindhoven, the Netherlands) operating at 120 kV, as described previously (Messner et al , 1986; Sekot et al , 2012). For non-invasive in vitro imaging by AFM using a Nanoscope III multimode AFM (Veeco Instruments Inc., Santa Barbara, CA), the sample was immobilized by mechanical trapping on a 0.8-μm polycarbonate membrane (Millipore) (Oh et al , 2013) and imaged in contact mode with a DNP-10 cantilever (Bruker, Vienna, Austria) with a nominal spring constant of 0.06 N m −1 and a nominal tip diameter of 20 nm. Scan line speed was 1 Hz.…”

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers

“…Unlike P. gingivalis that appears to have an amorphous surface layer composed of T9SS substrates, T. forsythia exhibits a true S-layer composed of two homologous T9SS substrates, TfsA and TfsB (Higuchi et al, 2000;Lee et al, 2006) that are important for various virulence traits (Sabet et al, 2003;Sakakibara et al, 2007). This S-layer has been studied by both atomic force microscopy and electron microscopy (EM) techniques and was determined to have a square (P4) lattice structure resulting from coassembly of equimolar amounts of TfsA and TfsB (Sekot et al, 2012;Oh et al, 2013). Homologs of the surface layer proteins are common in Tannerella and Parabacteroides species suggesting that the generation of T9SS-secreted surface layers may be common in this group of organisms.…”