Signaling pathways involved in inhibition of macrophage phagocytosis by TcpC of uropathogenic Escherichia coli
10.3760/cma.j.cn112309-20211115-00386
- VernacularTitle:尿路致病性大肠埃希菌通过TcpC抑制巨噬细胞吞噬的信号通路研究
- Author:
Xinpeng MA
1
;
Weiwei LUO
;
Wenting LI
;
Qian OU
;
Jialing QIU
;
Nuo CEN
;
Jiaqi FANG
Author Information
1. 浙大城市学院医学院，杭州 310015
- Keywords:
Uropathogenic Escherichia coli;
TIR domain-containing protein encoded by Escherichia coli (TcpC);
Ubiquitin ligase;
Inhibition;
Innate im
- From:
Chinese Journal of Microbiology and Immunology
2022;42(8):602-608
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the signaling pathway of inhibiting macrophage phagocytosis of TIR domain-containing protein encoded by Escherichia coli (TcpC) N-terminal ubiquitin ligase active fragments of uropathogenic Escherichia coli. Methods:Bioinformatics software was used to analyze the amino acid sequences and the function of TcpC N-terminal ubiquitin ligase active fragments as well as the functional sites. PCR was performed to amplify tcpc-330, tcpc-450 and tcpc-510 genes and a prokaryotic expression system was constructed to express the target proteins. The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were purified by Ni-NTA affinity chromatography. LPS in the recombinant proteins was removed by Detoxi-gel chromatography. The expression of MyD88 at protein and mRNA levels in macrophages incubated with rTcpC-N110, rTcpC-N150, rTcpC-N170 or rTcpC-TIR was detected by Western blot and qRT-PCR. The activation of NF-κB signal pathway and the levels of proinflammatory factors in macrophages incubated with the above TcpC protein fragments were measured by Western blot and ELISA, respectively. Results:Cys12, Trp104 and Trp106 in the N-terminal fragment of TcpC were crucial amino acids in maintaining its ubiquitin ligase activity. The target recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were successfully expressed and purified. After Detoxi-gel chromatography, rTcpC-N110, rTcpC-N150 and rTcpC-N170 extracts were undetectable for LPS. TcpC ubiquitin ligase fragments inhibited the expression of MyD88 at protein level, but not affect its expression at mRNA level in macrophages. LPS-induced phosphorylation of NF-κB signaling pathway-related proteins p50 and p65 was significantly inhibited in macrophages treated with TcpC ubiquitin ligase fragments. Moreover, LPS-induced production of pro-inflammatory factors was also significantly inhibited.Conclusions:The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 could inhibit the expression of MyD88 at protein level and suppress the activation of NF-κB signaling pathway, suggesting that they were closely related to the inhibition of innate immune activity of macrophages.
