Effects of Enolase of Streptococcus equi ssp. zooepidemicus on Phagocytic Functions of Alveolar Macrophages in Mice |
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| Authors: | CHEN Kaiwen HUA Chengwei YUAN Chen PAN Fei LIN Huixing FAN Hongjie MA Zhe |
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| Institution: | College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China |
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| Abstract: | To investigate the effect of enolase (Eno) of Streptococcus equi ssp. zooepidemicus (SEZ) on phagocytosis of mouse alveolar macrophages (RAW264.7). Recombinant enolase (rEno) was obtained by constructing prokaryotic expression plasmid, and the cytotoxicity of rEno protein on RAW264.7 cell proliferation was determined by trypan-blue living cell count method. After the rEno protein was incubated with RAW264.7 cells, SEZ was applied to the cells and the quantity of bacteria being phagocytosed was detected to determine the phagocytic activity of RAW264.7 cells. Further, candidate proteins that might interact with SEZ Eno in RAW264.7 cells were screened by live cell stable isotope labeling (SILAC) and protein spectrum analysis (LC-MS/MS). It was found that protein treatment (rEno，10 μg·mL-1) had significant cytotoxic effects on RAW264.7 cells. Treatment of RAW264.7 cells with 10.0 μg·mL-1 rEno protein for 2 and 4 hours could significantly inhibit the phagocytosis of RAW264.7 cells (P<0.01, P<0.05). In RAW264.7 cells, dynactin subunit protein 2 (Dctn), integrin alpha-M and about 17 proteins that might interact with Eno were preliminarily identified as rEno interaction proteins. The rEno recombinant expression protein was obtained in this study, and it could reduce the phagocytosis of RAW264.7 cells to SEZ. Preliminary screening of interacting proteins also laid a foundation for further revealing the mechanism of Eno in the anti-phagocytosis of SEZ. |
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| Keywords: | SEZ enolase RAW264 7 cell phagocytosis interactions between protein |
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