A Mycoplasma fermentans-derived Synthetic Lipopeptide Induces AP-1 and NF-κB Activity and Cytokine Secretion in Macrophages via the Activation of Mitogen-activated Protein Kinase Pathways

Abstract

Mycoplasma lipoproteins have been demonstrated to stimulate monocytic cells and induce proinflammatory cytokine secretion. In this paper, we show that a synthetic analog of the Mycoplasma fermentans membraneassociated lipopeptide macrophage-activating lipopeptide-2 (MALP-2) induces mRNA synthesis and protein secretion of interleukin-1␤ and tumor necrosis factor-␣ in human monocytes/macrophages and the murine macrophage cell line RAW 264.7, whereas the nonlipidated counterpart lacks this effect, underscoring the importance of protein acylation for cell activation. Synthetic MALP-2 (sMALP-2) induced the activation of MAPK family members extracellular signal regulated kinases 1 and 2, c-Jun NH 2 -terminal kinase, and p38 and induced NF-B and AP-1 transactivation in macrophages. Whereas the specific p38 inhibitor SB203580 abrogated both cytokine synthesis and NF-B and AP-1 transactivation in response to MALP-2, the selective MAPK/extracellular signal-regulated kinase-1 inhibitor PD-98059 decreased interleukin-1␤ and tumor necrosis factor-␣ production in response to sMALP-2 without affecting the transactivation of NF-B or AP-1. These results indicate that activation of MAPKs by sMALP-2 is a crucial event leading to the expression of proinflammatory cytokines. Our findings demonstrate that the synthetic analog of MALP-2 reproduces the macrophage stimulation activity found in different fractions of mycoplasmas. Given that MALP-2 has been recently shown to be expressed at the surface of M. fermentans as a molecular entity, sMALP-2 constitutes a valuable surrogate for investigating immunomodulation by these microorganisms and evaluating the role that this activity plays in the development of inflammatory diseases associated with mycoplasma infections.Mycoplasma fermentans, a human pathogen, is a potent activator of monocytes/macrophages. Macrophage activation by M. fermentans results in the secretion of numerous cytokines including interleukin (IL) 1 -1␤, IL-6, IL-8, IL-10, and tumor necrosis factor-␣ (TNF␣) (1-3). Other inflammation mediators such as NO have also been shown to be produced by macrophages in response to mycoplasmas (3). Mycoplasmas, the smallest self-replicating bacteria, are characterized by a wallless envelope (4); thus, they are LPS-free pathogens. We have previously demonstrated that mycoplasma membrane lipoproteins (lipid-associated membrane proteins; LAMPs) are responsible for human and murine macrophage activation (1, 2, 5).Although the signaling pathways triggered in macrophages by both M. fermentans LAMP and Gram-negative bacteria LPS are comparable, unlike LPS, M. fermentans LAMP activity does not require binding to CD14 or serum proteins (5). M. fermentans LAMP activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun NH 2 -terminal kinase (JNK), and p38 (5).MAPKs have been shown to be involved in M. fermentans LAMP-mediated cytokine induction, since selective inhibitors of these pathways have been shown to impair M. fermenta...

Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages toward YAC-1 tumor cells through production of NO

“…The mechanisms involved in NO and TNF induction by mycoplasmas are poorly understood. Recently, it was shown that mycoplasma activates the transcription factor NF-B in DMBM-3 macrophage cell line [15] and induces transactivation of NF-B and AP-1 in the Raw 264.7 macrophage cell line [14]. It was also reported that Mycoplasma fermentans induces protein tyrosine phosphorylation [31] and activation of the three subgroups of mitogenactivated protein kinases (MAPKs), ERK 1/2, JNKs, and MAPK p38, and that induction of TNF production by mycoplasma involves the ERK 1/2 and p38 kinases [13].…”

“…Its structure was elucidated and it constitutes one of the most potent macrophage stimulators [11] together with a lipopeptide recently isolated from M. hyorhinis membranes [12]. The characterization of mycoplasma-derived lipopeptides brought new insights into the mechanisms of NO and TNF induction in macrophages stimulated with mycoplasmas [13][14][15].…”

Response toNeisseria gonorrhoeaeby Cervicovaginal Epithelial Cells Occurs in the Absence of Toll-Like Receptor 4-Mediated Signaling

“…Synthetic lipopeptide corresponding to the N termini of the Treponema pallidum 47-kDa lipoprotein (designated 47-L) was a gift from J. Radolf (University of Connecticut, Farmington, CT). Synthetic lipopeptide based on the full-length MALP-2 membrane lipopeptide from Mycoplasma fermentans (sMALP-2) was a gift from G. Rawadi (Institut Pasteur, Paris, France) (27,42). Soluble peptidoglycan (designated sPG) was a gift from R. Dziarski (Indiana University School of Medicine, Gary, IN).…”

Cutting Edge: Preferentially the R-Stereoisomer of the Mycoplasmal Lipopeptide Macrophage-Activating Lipopeptide-2 Activates Immune Cells Through a Toll-Like Receptor 2- and MyD88-Dependent Signaling Pathway

“…Little is known about the signal pathways or the cell-surface receptors for MALP-2, except that MALP-2 activates the nuclear transcription factor NF-B (11,12). A new class of receptors of the innate immune system, the so-called Toll-like receptors (TLRs), was recently discovered (13)(14)(15), which recognize various bacterial cell-wall components such as LPS, peptidoglycan (PGN), LTA, and lipoproteins/lipopeptides (16 -22).…”