Biological characterization of lipopolysaccharide from Treponema pectinovorum

Abstract

This study investigated the endotoxic and biological properties of purified lipopolysaccharide (LPS) isolated from an oral spirochete, Treponema pectinovorum. Endotoxicity, measured by Limulus amoebocyte lysate kinetic assay, showed that the LPS contained 1.28 endotoxin units per microg of purified LPS, which was approximately 4,000 times less than Escherichia coli O55:B5 LPS. To determine in vivo endotoxicity, LPS responder mice were administered LPS following galactosamine (GalN) sensitization. The LPS induced neither endotoxic symptoms nor lethality for 96 h, suggesting negligible or very low endotoxicity. In contrast, infection with live T. pectinovorum induced 100% lethality within 12 h in GalN-sensitized LPS responder mice, indicating an endotoxin-like property of this treponeme. Heat-killed microorganisms exhibited no lethality in GalN-sensitized mice, suggesting that the endotoxicity was associated with heat-labile components. To determine cytokine and chemokine induction by LPS, human gingival fibroblasts were stimulated and secretion of interleukin 1beta (IL-1beta), granulocyte-macrophage colony-stimulating factor, gamma interferon, IL-6, IL-8, and monocyte chemoattractant protein 1 (MCP-1) was assessed. The purified LPS induced significant amounts of only IL-6, IL-8, and MCP-1, although they were substantially lower than levels after challenge with live T. pectinovorum. After injection of LPS or live or heat-killed T. pectinovorum, serum was collected from mice and analyzed for proinflammatory cytokines IL-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-6. LPS induced only IL-6 consistently. Both live and heat-killed T. pectinovorum induced serum IL-6, which was higher than the level detected following LPS administration. Importantly, live bacteria elicited systemic TNF-alpha and IL-1beta levels similar to those induced by a lethal dose of live E. coli O111. The results indicated that T. pectinovorum LPS has very low or no endotoxicity, although it can elicit low levels of cytokines from host cells. In contrast to the LPS, live T. pectinovorum demonstrated in vivo toxicity, which was associated with serum IL-1beta, TNF-alpha, and IL-6, suggesting an endotoxin-like property of a heat-labile molecule(s) of the spirochete.

FIG. 1.

IL-6, IL-8, and MCP-1 secretion by HGFs (Gin-7) challenged with purified T. pectinovorum ATCC 33768 LPS at 0.1, 1, 10, and 100 μg per ml. Supernatants were collected at 1, 3, 6, 12, 24, and 48 h after LPS challenge. Each cytokine concentration was determined by sequencial ELISA. The cells were stimulated with 5 × 10⁹ bacteria. The bars denote the mean level of cytokine detected during 12-and 24-h culture periods from triplicate determinations.

FIG. 2.

Cytokine (IL-1β, TNF-α, and IL-6) pattern in C3H/HeN Tac-MTV (LPS responder) mice after LPS treatment. Mice (n = 3) were injected i.p. with 5, 0.5, and 0.05 μg of T. pectinovorum ATCC 33768 LPS with GalN as indicated in Materials and Methods. Serum samples were taken at 3, 8, and 12 h after LPS administration. Each cytokine concentration was determined by ELISA. Results are the means and standard deviations for three mice at 12 h.

FIG. 3.

Proinflammatory-cytokine (IL-1β, TNF-α, and IL-6) patterns in C3H/HeN Tac-MTV (LPS responder) mice were analyzed after i.p. challenge infection with live (10⁸ cells) and heat-killed (H-K) (10⁸ cells) T. pectinovorum ATCC 33768 and live E. coli O111 (10⁵ cells) with and without GalN as described in Materials and Methods. Serum samples were taken at 3, 8, and 12 h postinfection. Each cytokine concentration was determined by sequential ELISA. Results are the means and standard deviations for three mice at 12 h.