Localization of Major, High Molecular Weight Proteins in <i>Bacteroides forsythus</i>

Higuchi, Naoya; Murakami, Yukitaka; Moriguchi, Keiichi; Ohno, Nobutada; Nakamura, Hiroshi; Yoshimura, Fuminobu

doi:10.1111/j.1348-0421.2000.tb02563.x

Cited by 26 publications

(37 citation statements)

References 14 publications

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“…S-layer-like proteins were purified from sonicated extract of B. forsythus ATCC 43037 according to the methods of Higuchi et al (10). In brief, the washed cells were sonicated on ice in the presence of protease inhibitors (0.25 mM N-␣-p-tosyl-L-lysine chloromethyl ketone, 0.2 mM phenylmethylsulfonyl fluoride, and 0.1 mM leupeptin; Sigma).…”

Section: Methodsmentioning

confidence: 99%

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Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

Yoneda

Hirofuji

Motooka

et al. 2003

Clin Vaccine Immunol

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Bacteroides forsythus is one of the important periodontopathic bacteria, and this microorganism is known to have an S-layer outside the outer membrane. The S-layer-like antigens were recently isolated from B. forsythus, and they were found to be 270-and 230-kDa proteins in the envelope fraction. In this study, these proteins were confirmed to be specific to B. forsythus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they were clearly recognized by sera from patients with adult and early-onset periodontitis in Western immmunoblot analysis. We compared the immunoglobulin G (IgG) responses against the purified S-layer-like antigen by enzyme-linked immunosorbent assay. IgG responses against this antigen were low in healthy control subjects, but they were significantly higher in subjects with adult and early-onset periodontitis. Together with the fact that the IgG responses against the crude extract of B. forsythus did not rise significantly in patients with periodontitis, S-layer-like proteins are considered to be specific antigens of B. forsythus and may play an important role in the progression of periodontitis.

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Section: Methodsmentioning

confidence: 99%

“…The functions of an S-layer of B. forsythus are as yet unknown, but Kerosuo suggests that S-layers may contribute to the rigidity of the cell wall (13). Recently, Higuchi et al reported that 270-and 230-kDa proteins in the envelope fraction of B. forsythus are constituents of the S-layer of this bacterium (10).…”

mentioning

confidence: 99%

Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

Yoneda

Hirofuji

Motooka

et al. 2003

Clin Vaccine Immunol

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“…Antiserum against the 270-kDa proteins was raised in a rabbit. The specificity of the antiserum was verified by Western blot analysis as described previously (Higuchi et al, 2000).…”

Section: Antigen Preparationmentioning

confidence: 99%

“…Envelope and soluble fractions were separated by ultracentrifugation of the whole cell extracts at 143,000 × g for 60 min as described previously (Higuchi et al, 2000). The envelope fraction was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Lugtenberg et al, 1975).…”

Section: Antigen Preparationmentioning

confidence: 99%

“…It was found that 230 and 270-kDa proteins might be the component of the S-layer, based upon the results of the immunoelectron microscopic study of 4% paraformaldehyde-0.05% glutaraldehyde fixation without OsO 4 or freeze-substituted method. (Higuchi et al, 2000;Moriguchi et al, 2003). Sabet et al (2003) reported that the S-layer consists of 200 and 210-kDa proteins.…”

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Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy

Moriguchi¹,

Jogahara²,

Kurihara³

et al. 2008

Okajimas Folia Anat. Jpn.

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In the electron microscopic immunocytochemistry, the general ultrastructure was sometimes sacrificed to obtain specific and efficient dense labeling. Antigenicity is often destroyed by the conventional post-fixation of tissue with OsO 4 (Roth et al., 1981). However, omission of OsO 4 results in poor membrane contrast and thereby compromises fine structure. Therefore, several investigations have used potassium ferrocyanide-reduced OsO 4 by which an excellent preservation of the ultrastructure and good immunoreactivity of the antigens became possible (Tamaki and Yamashina, 1994;Tamatani et al., 1995). Post-fixation by potassium ferrocyanide-reduced OsO 4 has been widely applied to obtain fine membrane structures with a clear contrast for morphological observation by transmission electron microscopy (Doine et al., 1984;Rambourg et al., 1993). However, we found that postfixed Tannerella forsythensis, a bacterium associated with periodontitis, followed by potassium ferrocyanidereduced OsO 4 showed no retention of immunocytochemical reactivity. It was found that 230 and 270-kDa proteins might be the component of the S-layer, based upon the results of the immunoelectron microscopic study of 4% paraformaldehyde-0.05% glutaraldehyde fixation without OsO 4 or freeze-substituted method. (Higuchi et al., 2000;Moriguchi et al., 2003). Sabet et al. (2003) reported that the S-layer consists of 200 and 210-kDa proteins. In their study, the bacterial cells were resuspended in 0.1 M cacodylate buffer with 1% glutaraldehyde, and post fixed with 1% OsO 4 . As optimal presentation of antibody binding activity, a large number of immunogold particles and their clear localization are an important requirement in the immunocyto-

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Energy dispersive spectroscopy‐scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non‐opsonized Tannerella forsythia

Moriguchi

Hasegawa

Higuchi

et al. 2017

Microscopy Res & Technique

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We investigated the association between human polymorphonuclear leukocytes (PMNs) and non-opsonized Tannerella forsythia ATCC 43037 displaying a serum-resistant surface layer (S-layer). When PMNs were mixed with T. forsythia in suspension, the cells phagocytosed T. forsythia cells. Nitro blue tetrazolium (NBT) reduction, indicative of O2- production, was observed by light microscopy; cerium (Ce) perhydroxide deposition, indicative of H O production, was observed by electron microscopy. We examined the relationship between high-molecular-weight proteins of the S-layer and Ce reaction (for T. forsythia phagocytosis) using electron microscopic immunolabeling. Immunogold particles were localized within the PMNs and on cell surfaces, labelling at the same Ce-reacted sites where the S-layer was present. We then used energy dispersive spectroscopy (EDS)-scanning transmission electron microscope (STEM) to perform Ce and nitrogen (N) (for S-layer immunocytochemistry) elemental analysis on the phagocytosed cells. That is, the elemental mapping and analysis of N by EDS appeared to reflect the presence of the same moieties detected by the 3,3'-diaminobenzidine-tetrahydrochloride (DAB) reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies, instead of immunogold labeling. We focused on the use of EDS-STEM to visualize the presence of N resulting from the DAB reaction. In a parallel set of experiments, we used EDS-STEM to perform Ce and gold (Au; from immunogold labeling of the S-layer) elemental analysis on the same phagocytosing cells.

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Localization of Major, High Molecular Weight Proteins in Bacteroides forsythus

Cited by 26 publications

References 14 publications

Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

Humoral Immune Responses to S-Layer-Like Proteins of Bacteroides forsythus

Immunocytochemical approach for surface layer proteins of freeze-substituted Tannerella forsythensis by energy-filtering transmission electron microscopy

Energy dispersive spectroscopy‐scanning transmission electron microscope observations of free radical production in human polymorphonuclear leukocytes phagocytosing non‐opsonized Tannerella forsythia

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