[Zinc-dependent metalloprotease 1 (Zmp1) inhibits the proliferation and phagosome-lysosome fusion of mouse macrophage RAW264.7].
Objective To observe the effect of zinc-dependent metalloprotease 1 (Zmp1) of Bacillus Calmette Guerin vaccine (BCG) on the proliferation and phagosome-lysosome fusion of RAW264.7 mouse macrophage. Methods Zmp1 was expressed in E.coli BL21 (DE3) and purified by metal chelate magnetic beads. RAW264.7 cells were incubated with the purified Zmp1. Cell proliferation was detected at 0, 24, 48, 72 and 96 hours by cell counting kit-8 (CCK-8) assay and cell cycle distribution was detected by flow cytometry (FCM). The formation of phagosome was induced after RAW264.7 cells were infected with attenuated Salmonella. Attenuated Salmonella in phagosomes and lysosome associated membrane protein 1 (LAMP1) were marked by Alexa FluorR350-anti-Salmonella antibodies (blue fluorescence) and Cy5-anti-LAMP1 antibodies (red fluorescence), respectively. Two kind of fluorescence in RAW264.7 cells were observed under a fluorescent microscope and the fusion of phagosome and lysosome was analyzed by overlaying two kind of fluorescence (purple fluorescence). Results RAW264.7 cell proliferation decreased obviously 48 hours after treated with Zmp1, and the cell count in G1 phase declined, but the number of S-phase cells increased. In RAW264.7 cells infected with attenuated Salmonella and labeled by double immunofluorescent staining, phagolysosomes exhibiting purple fluorescence were reduced after treated by Zmp1. Conclusion Zmp1 can inhibit mouse RAW264.7 cell proliferation and phagosome-lysosome fusion.
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chonqing Medical University, Chongqing 400016, China.
Chencheng
C
Fang
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chonqing Medical University, Chongqing 400016, China.
Jiming
J
Zhang
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chonqing Medical University, Chongqing 400016, China.
Nan
N
Lu
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chonqing Medical University, Chongqing 400016, China.
Peng
P
Li
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chonqing Medical University, Chongqing 400016, China.
Yu
Y
Fan
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chonqing Medical University, Chongqing 400016, China.
Yuexi
Y
Kang
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chonqing Medical University, Chongqing 400016, China. *Corresponding authors, E-mail: heyonglinhyl@163.com.
Chun
C
Yang
Center for Molecular Medicine and Tumor Research, Department of Pathogen Biology, School of Basic Medicine, Chonqing Medical University, Chongqing 400016, China. *Corresponding authors, E-mail: yangchunim@163.com.
Yonglin
Y
He
D000818:Animals; D001500:BCG Vaccine / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects*; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology*; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects; D008561:Membrane Fusion / Q000187:drug effects*; D045726:Metalloproteases / Q000494:pharmacology*; D051379:Mice; D010588:Phagosomes / Q000187:drug effects / Q000502:physiology*; D000067996:RAW 264.7 Cells
D001500:BCG Vaccine; D001426:Bacterial Proteins; D045726:Metalloproteases