Inflammatory lipoproteins purified from a toxigenic and arthritogenic strain of Mycoplasma arthritidis are dependent on Toll-like receptor 2 and CD14

Abstract

Mycoplasma arthritidis is a naturally occurring murine pathogen, and the disease model has been used extensively to understand inflammatory mechanisms. Recently, Triton X-114 extracts of a virulent strain of M. arthritidis were found to be more potent in activating macrophages than were those from an avirulent strain, suggesting a role in disease. Here, octyl glucoside extraction of cells was used to identify four distinct bioactive moieties, with molecular masses of approximately 41, 37, 34, and 17 kDa. Their bioactivities were resistant to proteinase K but were destroyed by alkaline hydrolysis and oxidation. As for MALP-2, all were dependent upon Toll-like receptor 2, but unlike MALP-2, they were also dependent upon CD14. The M. arthritidis lipoproteins exhibited infrared absorbances at 2,900 cm(-1) and 1,662 cm(-1), similar to those seen in Pam(3)-Cys-Ser-(Lys)(4). Edman degradation failed to reveal N-terminal sequences, suggesting that they were blocked and therefore might be triacylated. However, mass spectrometry of fragments revealed that the 41-kDa moiety, which binds to serum apolipoprotein A-1, had similarity with the recently described MlpD lipoprotein of M. arthritidis.

FIG. 1.

Purification of bioactive components from M. arthritidis. The following purification data are representative of repeated OG extractions on two separate batches of organisms. (A) Dose dependency of OGex induction of TNF-α in murine RAW 264.7 cells. (B) SDS-PAGE of OGex. Lane 1, protein standards; lane 2, OGex. The gel was blotted to nitrocellulose; 2-mm strips were excised, dissolved in dimethyl sulfoxide, and precipitated; and the (lipo)protein-coated particles were tested for TNF-α production in culture supernatants of RAW cells. (C) SDS-PAGE of purified lipoproteins stained with silver. Lane 1, protein standards; lane 2, OGex A; lane 3, OGex B; lane 4, OGex C; lane 5, OGex D. (D) Dose response of TNF-α production by purified OGex A, B, C, and D in RAW 264.7 cells.

FIG. 2.

Properties of OGex. (A) SDS-PAGE of OGex. Lane 1, protein standards; lane 2, OGex treated with PK. PK-treated OGex were blotted to cellulose nitrate strips, extracted, and tested for activity, all of which migrated to the dye front. (B) OGex were incubated with NS or with PK and, after being heated to 100°C for 15 min, were assayed for the ability to induce TNF-α in RAW cell cultures. (C) Effect of alkaline hydrolysis on the activity of 50 ng/ml of OGex A, B, C, and D, as indicated by the ability to stimulate TNF-α production by RAW 264.7 cells. (D) Effect of H₂O₂ treatment on bioactivity of OGex A, B, C, and D. (C and D) Tests for lipid groups were carried out on three separate cell suspensions stimulated with each of the treated OGex. Mean results ± standard deviations are shown.

FIG. 3.

IR spectra of Pam₃CSK₄ and OGex A, B, C, and D. White arrows show the signals at about 2,900 cm⁻¹, suggesting the presence of fatty acid acyl chains. Black arrows show the signals at about 1,700 cm⁻¹, suggesting the presence of ester bonds.

FIG. 4.

Flow cytometric analysis of CHO/TLR2 cells. The cells were stained with fluorescein isothiocyanate-labeled anti-human CD25 MAb and analyzed by flow cytometry for the expression of the CD25 transgene, an indicator of TLR activation. The activation of cells was expressed by mean fluorescence intensity (MFI). Thin lines, no antibody; broken lines, isotype control antibody; thick lines, anti-human CD25 MAb.

FIG. 5.

(A) TNF-α production by peritoneal macrophages of C57BL/6 mice or C57BL/6 TLR2 KO mice stimulated with LPS, NS, or OGex A, B, C, or D. The concentration of LPS and OGex A, B, C, and D was 100 ng/ml. Peritoneal adherent cells were stimulated for 18 h with inducers, and the amounts of TNF-α in cell culture supernatants were tested by ELISA. The data are representative of two experiments using four to five mice each. The experiment was repeated twice. (B) RAW 264.7 cells were preincubated with NS, isotype control antibody (Ab), or anti-mouse CD14 MAb (10 μg/ml) for 1 h. Then, they were stimulated with LPS (10 ng/ml), MALP-2, or OGex A, B, C, or D (20 ng/ml) for 18 h. (C) Peritoneal macrophages from C57BL/6 or C57BL/6 CD14 KO mice were stimulated with 50 ng/ml LPS, MALP-2, or OGex A, B, C, or D, and culture supernatants were tested for the presence of TNF-α. Representative data from two similar experiments using four to five mice each are shown.

FIG. 6.

Amino acid sequence comparisons between OGex A fragments and MlpD, MlpE, and MlpF. Amino acid residues identical to one or both of the OGex A peptides are shaded black.

FIG. 7.

Dot blot analysis of OGex A, B, C, and D. Recombinant MlpA, MlpC, MlpD, MlpE, and MlpF lipoproteins and OGex A, B, C, and D were placed on a PVDF membrane and exposed to anti-Mlp antiserum. (A) Schema of protein spotting patterns. (B) PVDF membranes of dot blot analysis. Antigens were detected by use of anti-Mlp serum. The amount of protein used was 2 μg/dot. The experiment was conducted three times using different antigen concentrations but with similar results. rMlp, recombinant Mlp.