Mammalian cell entry protein of Mycobacterium tuberculosis induces the proinflammatory response in RAW 264.7 murine macrophage-like cells.
Mammalian cell entry (Mce1A) protein of Mycobacterium tuberculosis (Mtb) is involved in bacterial entry and survival in macrophages, which has been shown to induce production of tumor necrosis factor alpha (TNF-alpha). It remains unclear whether and how Mce1A functions upon the type I interleukin-1 receptor-associated protein kinase (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) of important proinflammatory cytokines. In this study, His-tagged Mce1A was expressed and purified. Also, two pieces of small interfering RNA (siRNA) were designed and synthesized by in vitro transcription, which exhibit specific and efficient silencing effect on mce1a expression. Furthermore, RAW 264.7 murine macrophage-like cells were exposed to His-tagged Mce1A or co-transfected with the Mce1A-expressing plasmid and efficient siRNA, and levels of IRAK-1 and TRAF-6 were then determined by Western blot. We show here that Mce1A induces up-regulations of IRAK-1 and TRAF-6 in macrophages in a dose-dependent manner. The level of Caspase-3 closely related with apoptosis was also determined, whereas no changes were observed. These results indicate that Mtb Mce1A protein induces a proinflammatory response in macrophages.
Xue
Li-Jun
LJ
Department of Microbiology, State Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai 200433, PR China.
Cao
Ming-Mei
MM
Luan
Jie
J
Ren
Hao
H
Pan
Xin
X
Cao
Jie
J
Qi
Zhong-Tian
ZT
D000818:Animals; D001426:Bacterial Proteins / Q000276:immunology*; D015153:Blotting, Western; D053148:Caspase 3 / Q000032:analysis; D002478:Cells, Cultured; D066298:In Vitro Techniques; D053592:Interleukin-1 Receptor-Associated Kinases / Q000032:analysis*; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000502:physiology*; D048029:TNF Receptor-Associated Factor 6 / Q000032:analysis*; D014158:Transcription, Genetic; D015854:Up-Regulation / Q000502:physiology
D001426:Bacterial Proteins; D048029:TNF Receptor-Associated Factor 6; C473802:mammalian cell entry protein Mce1A, Mycobacterium tuberculosis; D053592:Interleukin-1 Receptor-Associated Kinases; D053148:Caspase 3