RESUMO
In addition to regulating B cell development and activation, Bruton's tyrosine kinase (Btk) functions downstream of multiple TLRs, including TLR7, to regulate innate immune responses in myeloid cells. Although critical for defense against RNA viruses such as influenza and Sendai virus, recognition of self-RNA by TLR7 also has been shown to be an important contributor to the pathophysiology of systemic lupus erythematosus. To date, the role of Btk in regulating TLR7-mediated responses is poorly understood. In the current study, we have demonstrated a hitherto undiscovered role for Btk in apoptotic cell uptake, identifying the molecular chaperone calreticulin (CRT) as a novel substrate for Btk in regulating this response. CRT together with the transmembrane receptor CD91 function at the cell membrane and regulate uptake of C1q-opsonised apoptotic cells. Our results show that Btk directly phosphorylates CRT and that in the absence of Btk, CRT fails to localize with CD91 at the cell surface and at the phagocytic cup. Critically, a blocking Ab against CRT in wild-type macrophages mimics the inability of Btk-deficient macrophages to phagocytose apoptotic cells efficiently, indicating the critical importance of Btk in regulating CRT-driven apoptotic cell uptake. Our data have revealed a novel regulatory role for Btk in mediating apoptotic cell clearance, with CRT identified as the critical component of the CRT/CD91/C1q system targeted by Btk. Given the importance of clearing apoptotic cell debris to prevent inappropriate exposure of TLRs to endogenous ligands, our results have important implications regarding the role of Btk in myeloid cell function.
Assuntos
Apoptose , Calreticulina/metabolismo , Complemento C1q/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor 7 Toll-Like/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Anticorpos Bloqueadores/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Ativação Linfocitária , Macrófagos/imunologia , Proteínas de Membrana , Camundongos , Células Mieloides/imunologia , Fagocitose/imunologia , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Receptores de LDL/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismoRESUMO
The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections, including chronic biofilm infections in the lungs of individuals with cystic fibrosis. We previously found that the inner-membrane protein MgtE can function both as a magnesium transporter and a virulence modulator, although the exact mechanism governing these activities is unclear. To address this issue, we carried out an experimental characterization of P. aeruginosa MgtE and generated a computer-rendered model. Our in silico analysis demonstrated the structural similarity of P. aeruginosa MgtE to that of the crystal structure of MgtE in Thermus thermophilus. Experimentally, we verified that MgtE is not essential for growth and found that it may not be involved directly in biofilm formation, even under low-magnesium conditions. We demonstrated both magnesium transport and cytotoxicity-regulating functions, and showed that magnesium-binding sites in the connecting helix region of MgtE are vital in coupling these two functions. Furthermore, limiting magnesium environments stimulated mgtE transcriptional responses. Our results suggested that MgtE might play an important role in linking magnesium availability to P. aeruginosa pathogenesis.
Assuntos
Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Magnésio/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Pseudomonas aeruginosa/fisiologia , Antiporters/química , Antiporters/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Sobrevivência Celular , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Deleção de Genes , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Thermus thermophilus/químicaRESUMO
The microtiter plate (also called 96-well plate) assay for studying biofilm formation is a method which allows for the observation of bacterial adherence to an abiotic surface. In this assay, bacteria are incubated in vinyl "U"-bottom or other types of 96-well microtiter plates. Following incubation, planktonic bacteria are rinsed away, and the remaining adherent bacteria (biofilms) are stained with crystal violet dye, thus allowing visualization of the biofilm. If quantitation is desired, the stained biofilms are solubilized and transferred to a 96-well optically clear flat-bottom plate for measurement by spectrophotometry.