Mapping of the proinflammatory domains of MspTL of Treponema lecithinolyticum.
The major surface protein (MspTL) of Treponema lecithinolyticum, associated with periodontitis and endodontic infections, has been reported to induce proinflammatory mediators such as intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-1beta, IL-6 and IL-8. The purpose of this study was to examine the role of MspTL in cell adhesion/migration and to identify its proinflammatory domains. Using the human monocytic cell line THP-1 and human dermal microvascular endothelial cells (HMEC-1), it was demonstrated that MspTL increased adhesion of monocytes to endothelial cells and transendothelial migration. To analyse the proinflammatory domains of the protein, four gene constructs covering different regions of MspTL were designed and expressed in Escherichia coli using the expression vector pQE-30. Histidine-tagged recombinant proteins were purified using Ni-NTA agarose and polymyxin B agarose to remove LPS contamination. Recombinant truncated polypeptides were assessed for the ability to induce ICAM-1 and proinflammatory factors in THP-1 cells by real-time RT-PCR and ELISA. Of the four polypeptides, the one spanning the N-terminal 86 amino acids significantly induced ICAM-1, IL-1beta, IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha), cyclooxygenase (COX)-2, and prostaglandin E2 (PGE2). The results indicate that MspTL may induce cell adhesion and inflammation via its N-terminal region.
Jun
Hye-Kyoung
HK
Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.
Lee
Hae-Ri
HR
Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.
Lee
Sung-Hoon
SH
Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.
Choi
Bong-Kyu
BK
Dental Research Institute, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.|Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis*; D015232:Dinoprostone / Q000096:biosynthesis; D042783:Endothelial Cells / Q000382:microbiology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009000:Monocytes / Q000276:immunology; D018272:Porins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014208:Treponema / Q000235:genetics / Q000276:immunology* / Q000502:physiology
D001426:Bacterial Proteins; D016207:Cytokines; D018272:Porins; D011994:Recombinant Proteins; C099361:major outer sheath protein, Treponema; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone