Toshihiko Umemoto

Treponema medium sp. nov., Isolated from Human Subgingival Dental Plaque

A new Treponema species, for which we propose the name Treponema medium, was isolated from subgingival plaque from an adult with periodontal disease. The morphological characteristics, differential biochemical characteristics, and protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of this organism are described. The guanine-plus-cytosine content of the DNA of T. medium is 51 mol%. The levels of DNA-DNA relatedness of the new species to other Treponema species, including Treponema denticola, Treponema vincentii, Treponema socranskii, Treponema pallidum, and Treponema phagedenis, are less than 30%. A phylogenetic analysis based on 16S rRNA sequences distinguished the new Treponema strain from strains belonging to previously described Treponema species. The type strain of T. medium is strain G7201.

Fibronectin-Binding Proteins of a Human Oral Spirochete Treponema denticola

Major polypeptides from a human oral spirochete Treponema denticola ATCC 33520 were examined to demonstrate their ability to bind to human plasma fibronectin by immunoblot analysis. Of three main polypeptides separated on sodium dodecyl sulfate polyacrylamide gels 53,000‐daltons (53‐kDa) and 72‐kDa surface antigenic proteins and a 38‐kDa axial flagellar protein showed the ability to bind to fibronectin, suggesting that fibronectin on host cells can mediate cytoadherence of T. denticola by its binding to the surface proteins or the exposed 38‐kDa axial flageller protein.

Binding of host-associated treponeme proteins to collagens and laminin: a possible mechanism of spirochetal adherence to host tissues.

The polypeptides of seven strains of human treponemes were investigated by immunoblot analysis for their binding to the human placental collagens and laminin. Of the treponemal polypeptides, eleven polypeptides, 45‐kDa, 49‐kDa, and 62‐kDa polypeptides from T. pallidum ATCC 27087, a 48‐kDa polypeptide from T. phagedenis biotype Reiter, 51‐kDa and 53‐kDa polypeptides from T. vincentii ATCC 35580, 30‐kDa, 53‐kDa and 63‐kDa polypeptides from T. socranskii subsp. buccale ATCC 35534, a 52‐kDa polypeptide from T. denticola ATCC 35405, and a 53‐kDa polypeptide from T. denticola ATCC 33520 possessed an ability to bind to the laminin, type I, III, IV, or V collagen. An intermediate‐sized human oral isolate strain G7201 did not possess any laminin‐ or collagen‐binding polypeptides. Immunoelectron microscopy using intact treponemal cells with a single collagen‐binding polypeptide and the corresponding antisera demonstrated that the 51‐kDa and 53‐kDa polypeptides from T. vincentii, the 53‐kDa polypeptide from T. socranskii subsp. buccale ATCC 35534 and the 52‐kDa polypeptide from T. denticola ATCC 35405, were outer envelope proteins.

Adherence of Human Oral Spirochetes by Collagen-Binding Proteins

The ability of spirochetes to adhere to collagens was compared among three species of human oral treponemes. Immunoblot analysis demonstrated that type I‐, IV‐, and V‐collagen‐binding polypeptides (CBPs) were detected in the heated and unheated preparations from both Treponema denticola ATCC 33520 and T. socranskii subsp. buccale ATCC 35534. Few CBPs, however, were detected in the heated and unheated preparations from a recently characterized isolate, T. medium strain G7201. Immunoelectron microscopy using rabbit antisera against the CBPs from the unheated preparations demonstrated that four CBPs, a 27 kDa type V‐CBP of T. denticola ATCC 33520, a 95 kDa type IV‐CBP and a 110 kDa type I‐CBP of T. socranskii subsp. buccale ATCC 35534, and a 95 kDa type IV‐CBP of T. medium strain G7201, were located on the outer envelopes of the individual cells. The adherence of T. denticola to the collagen‐coated surfaces was significantly greater than that of T. medium, suggesting that the CBPs on the oral spirochetal cells play an important role in their adherence to collagen‐rich connective tissues of the host.

Bactericidal effect of rat cystatin S on an oral bacterium Porphyromonas gingivalis

We tested antibacterial and antiviral activities of rat cystatin S, a cysteine proteinase inhibitor, belonging to the family 2 cystatins against 18 different bacterial species and poliovirus type 1 (Sabin). Rat cystatin S specifically inhibited the growth of a human oral anaerobic bacterium Porphyromonas gingivalis due to a bactericidal effect.

Electron Microscopy of the Spherical Body of Oral Spirochetes In Vitro: Further Studies

The surface and inner structure of the spherical bodies (SB) produced by the human oral treponeme strain G7201, similar to Treponema macrodentium, were studied by electron microscopy. Ultrathin sectioning and scanning techniques demonstrated that in the presence of a high concentration of sucrose, the outer envelope of one or both terminal ends of this oral spirochete changed into a swollen structure, the SB. Spirochetal cells adhered firmly to the surface of the resultant body. The membrane of the SB, i.e. the outer envelope, enclosed the coiled protoplasmic cylinder and five axial fibrils which were located between the envelope and the cylinder. Large expanded protoplasmic cylinders were observed, surrounded by a partially disrupted double membrane in some SBs. A number of frizzly fibrous structures, which differed from axial fibrils in number and shape, were also observed within these SBs. Except for abnormal or partially broken cylinders, the protoplasmic cylinders tended to be located close to the inner surface of the SB membrane, resulting in a central vacant space with occasional axial fibrils.

Chemotaxis of Oral Treponemes toward Sera and Albumin of Rabbit

The motility and chemotaxis of human oral spirochetes Treponema denticola ATCC 35404, T. medium ATCC 700293, and T. vincentii ATCC 35580 were examined by a capillary assay method. Of five sera three human oral treponemes were dominantly chemoattractant to the rabbit serum. The checkerboard analysis of chemotaxis toward rabbit serum clearly showed that the motile T. denticola cells swam toward the culture media containing higher concentrations of the rabbit serum. T. denticola chemotaxis to the rabbit serum was clearly reduced by heating serum, and rabbit albumin contributed by 60 to 70% to its chemotaxis to the rabbit serum. Western blotting analysis demonstrated that these treponemes possessed rabbit albumin‐binding polypeptides with approximate molecular sizes of 65 kDa and 70 kDa. Immunoelectron microscopy demonstrated that a 65 kDa rabbit albumin‐binding polypeptide was located on the outer envelopes, suggesting that the rabbit albumin‐binding polypeptide is responsible for chemotaxis toward rabbit serum.

Fimbria‐Mediated Coaggregation between Human Oral Anaerobes Treponema medium and Porphyromonas gingivalis

Bacterial binding phenomena among different bacterial genera or species play an important role in bacterial colonization in a mixed microbiota such as in the human oral cavity. The coaggregation reaction between two gram‐negative anaerobes, Treponema medium and Porphyromonas gingivalis, was characterized using fimbria‐deficient mutants of P. gingivalis and specific antisera against purified fimbriae and bacterial whole cells. T. medium ATCC 700273 strongly coaggregated with fimbriate P. gingivalis strains ATCC 33277 and 381, but not with afimbriate strains including transposon‐induced fimbria‐deficient mutants and KDP98 as a fimA‐disrupted mutant of P. gingivalis ATCC 33277. In the P. gingivalis‐T. medium coaggregation assay, the presence of rabbit antiserum against the purified fimbriae or the whole cells of P. gingivalis ATCC 33277 produced different “aggregates” consisting predominantly of P. gingivalis cells with few spirochetes, but both preimmune serum and the antiserum against the afimbriate KDP98 cells did not inhibit the coaggregation reaction. Heated P. gingivalis cells lost their ability to bind both heated and unheated T. medium cells. This T. medium‐P. gingivalis coaggregation reaction was inhibited by a cysteine proteinase inhibitor, leupeptin, and also by arginine and lysine, but not by EDTA or sugars including lactose. A binding assay on nitrocellulose membranes and immunoelectron microscopy demonstrated that a heat‐stable 37 kDa surface protein on the T. medium cell attached to the P. gingivalis fimbriae.

Colonial Morphology of Treponemes Observed by Electron Microscopy

The colonial morphology of three strains of cultivable, nonpathogenic treponemes including a human oral treponeme was examined by light and electron microscopy. Treponema phagedenis strains Kazan and Reiter produced large white colonies on the surface of solid media composed of sterility test broth, 0.9 to 3.1% agar, rifampin, and 12.5% rabbit or horse serum. A human oral treponeme, strain G7201, grew as diffused white zones on 0.9 to 3.1% agar plates. Under the cultural conditions employed agar concentrations slightly affected the time of appearance of colonies of the three strains of treponemes.

Antigenic Behaviors of Two Axial Flagellar Proteins Detected in Treponema denticola

Two polypeptide antigens with molecular sizes of 34,000 daltons (34 kDa) and 38 kDa were separated from heated cells of a human clinical treponeme strain G7201 and Treponema denticola ATCC 35404, respectively. The rabbit polyclonal antisera against these antigens were produced and examined for their immunological reactions with the two heated antigens or intact spirochetal cells. Immunoblot analysis showed that the 34‐kDa protein was also detected in T. denticola ATCC 35404 and ATCC 33520, and the 38‐kDa protein was detected only in the two ATCC strains. Immunoelectron microscopy using the two rabbit antisera and protein A‐gold complexes demonstrated that the 38‐kDa protein antigen was present on the axial flagella of two T. denticola strains, and that the 34‐kDa protein was located in the axial flagella of the G7201 cell, but neither in axial flagella nor on outer envelopes of the two ATCC strains cells, suggesting that the native 34‐kDa axial flagellar protein of the G7201 strain may be different from that of T. denticola in terms of immunological reactivity.