A 4.1-kilodalton polypeptide in the cultural supernatant of Mycoplasma fermentans is one of the substances responsible for induction of interleukin-6 production by human gingival fibroblasts

Abstract

The cultural supernatant of Mycoplasma fermentans induced interleukin-6 production by human gingival fibroblasts. The active entities were divided into hydrophilic and hydrophobic substances. In this study, we purified a 4.1-kilodalton polypeptide from the hydrophilic substances. It reacted with polyclonal antibodies to M. fermentans and activated human macrophages.

FIG. 1

Growth of M. fermentans in liquid medium and IL-6 production-inducing activity in the cultural supernatant. M. fermentans was grown in liquid medium, and a 2-ml aliquot of the culture was taken periodically and divided into two portions. One portion was used to determine the pH (○) and the number of viable cells (CFU/ml, □). The other was centrifuged at 100,000 × g for 1 h to separate the cultural supernatant, which was used to determine the ability to induce IL-6 production by HGF (●).

FIG. 2

Protein profile (solid line) obtained by reversed-phase chromatography of P60 on an HPLC column. P60 was applied to a preparative Nucleosil 120-7 C₁₈ column (10 by 300 mm; Chemco Scientific Co. Ltd.). Fractionation was carried out by using the following program: at time zero, 5% DMF–95% MQW; at 15 min, 5% DMF–95% MQW; at 60 min, 5% DMF–95% 2-propanol; and at the end, 5% DMF–95% 2-propanol. The flow rate was 1.0 ml/min. Each fraction was dried in vacuo at 45°C, dissolved in MQW, and examined for IL-6 production-inducing activity (□). Abs, absorbance.

FIG. 3

SDS-PAGE. SDS-PAGE of polypeptide standards (A; 4.0 μg of protein) and peak C (B; 0.3 μg of protein) was performed with a 16% polyacrylamide gel. Proteins were stained by using a silver stain plus kit (Bio-Rad).

FIG. 4

Detection of substances reacted with anti-M. fermentans serum. M. fermentans cells (A, 4 μg of protein; B, 0.4 μg of protein) and peak C (C, 0.06 μg of protein) were spotted onto two nitrocellulose membranes. Each of the membranes was blocked with 5% skim milk in phosphate-buffered saline. One membrane was then reacted with anti-M. fermentans serum, and the other was reacted with preimmune serum. Each of antibodies was detected by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G and a DAB substrate (Vector Laboratories, Inc.).