Activation of inflammasomes in dendritic cells and macrophages by Mycoplasma salivarium.

Interleukin-1ß (IL-1ß) plays crucial roles in the pathogenesis of periodontal disease. It is produced after the processing of pro-IL-1ß by caspase-1, which is activated by the inflammasome-a multiprotein complex comprising nucleotide-binding domain leucine-rich repeat-containing receptor (NLR), the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and procaspase-1. Mycoplasma salivarium preferentially inhabits the gingival sulcus and the incidence and number of organisms in the oral cavity increase significantly with the progression of periodontal disease. To initially clarify the association of this organism with periodontal diseases, this study determined whether it induces IL-1ß production by innate immune cells such as dendritic cells or macrophages by using Mycoplasma pneumoniae as a positive control. Both live and heat-killed M. salivarium and M. pneumoniae cells induced IL-1ß production by XS106 murine dendritic cells as well as pyroptosis. The activities were significantly downregulated by silencing of caspase-1. Bone-marrow-derived macrophage (BMMs) from wild-type and NLR-containing protein 3 (NLRP3)-, ASC-, and caspase-1-deficient mice were examined for IL-1ß production in response to these mycoplasmas. Live M. salivarium and M. pneumoniae cells almost completely lost the ability to induce IL-1ß production by BMMs from ASC- and caspase-1-deficient mice. Their activities toward BMMs from NLRP3-deficient mice were significantly but not completely attenuated. These results suggest that live M. salivarium and M. pneumoniae cells can activate several types of inflammasomes including the NLRP3 inflammasome. Both M. salivarium and M. pneumoniae cells can activate THP-1 human monocytic cells to induce IL-1ß production. Hence, the present finding that M. salivarium induces IL-1ß production by dendritic cells and macrophages may suggest the association of this organism with periodontal diseases.

In situ immunohistochemical detection of intracellular Mycoplasma salivarium in the epithelial cells of oral leukoplakia.

BACKGROUND: Mycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia is the most prevalent potentially malignant disorder of the oral mucosa; its etiology has not been defined. Our previous study with DNA-binding fluorescent dye suggested the presence of mycoplasmas in the epithelial cells of leukoplakia tissue. OBJECTIVE: Our aim was to detect M. salivarium in the epithelial cells of leukoplakia by immunohistochemistry. DESIGN: We produced a polyclonal antibody (PAb) reactive to Mycoplasma by injecting a rabbit with M. salivarium cells (ATCC 23064) mixed with complete Freund's adjuvant and a monoclonal antibody specific to M. salivarium by injecting M. salivarium cells (ATCC 23557) mixed with complete Freund's adjuvant into the footpads of a rat. Then, we attempted to detect M. salivarium in the epithelium of leukoplakia tissues by immunohistochemistry. RESULTS: We obtained an antimycoplasma rabbit PAb reactive to all seven Mycoplasma species used in this study. Three hybridoma clones producing monoclonal antibodies specific to M. salivarium were obtained, and an M. salivarium-specific monoclonal antibody, designated 7-6H, was established. Immunohistochemistry with these antibodies revealed M. salivarium in the epithelial cells of leukoplakia with hyperplasia and hyperkeratosis on histology. PCR and sequencing verified the presence of M. salivarium DNA in the epithelial cells of leukoplakia. CONCLUSION: Intracellular M. salivarium was identified in the epithelial cells of leukoplakia.

Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6.

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam(2)CGDPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentans-derived lipopeptide MALP-2 (Pam(2)CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-kappaB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-kappaB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.