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.
ABSTRACT
Objective:To investigate whether the hypervirulent Klebsiella pneumoniae (hvKP) induces liver abscess through activating NLRP3 inflammasome. Methods:K1-hvKP and K35-non-hvKP bacterial suspensions were intraperitoneally injected into C57BL/6 mice to establish the models of liver abscess. Human peripheral blood neutrophils were sorted by immunomagnetic beads with CD45 + and Gr-1 + , and the purity was detected by flow cytometry. The concentrations of capsular polysaccharide of K1-hvKP and K35-non-hvKP were detected by total carbohydrate assay kit. The expression of IL-18 and IL-33 by neutrophils at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and ELISA, respectively. The activation of NLRP3 inflammasome in neutrophils was detected by Western blot. Neutrophil extracellular trap formation (NETosis) was observed under confocal laser scanning microscope. Results:The C57BL/6 mice with K1-hvKP infection had significantly serious liver abscess as compared with the K35-non-hvKP-infected mice. The purity of human neutrophils was more than 95%. The concentration of capsular polysaccharide in K1-hvKP was significantly higher than that in K35-non-hvKP. Compared with K35-non-hvKP, K1-hvKP significantly promoted the neutrophils to express IL-18 and IL-33 at both mRNA and protein levels, enhanced the activation of NLRP3 and induced NETosis.Conclusions:This study suggested that hvKP could promote NETosis by activating NLRP3 inflammasome to cause liver abscess.
ABSTRACT
Objective:To investigate the role of TcpC in uropathogenic Escherichia coli (UPEC)-induced cystitis in mice and to preliminarily analyze the pathogenic mechanism. Methods:C57BL/6 mice were injected with 10 9 CFU wild-type UPEC CFT073 (CFT073 wt) or tcpc gene-deleted mutant (CFT073 Δ tcpc) from urethra into bladder to construct the mouse model of cystitis. The mice were sacrificed 3 d after infection and the bladders were taken to observe the gross pathological changes. Histopathological changes in bladder tissues were observed after HE staining. Immunohistochemistry was used to detect TcpC in bladder tissues. Bacterial loads in urine samples of UPEC-infected mice were counted by tenfold dilution method, and the presence of tcpc gene in the genomic DNA of bacteria from the bladder and urine samples of CFT073 wt-infected mice was measured by PCR. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to detect the expression of TcpC at mRNA and protein levels in macrophages after CFT073 wt infection. The influence of UPEC strains on the activation of NF-κB signaling pathway in macrophages were determined by Western blot. The levels of proinflammatory factors and the bacterial and cell activity after infecting macrophages with UPEC strains were detected by ELISA, laser confocal microscope and fluorescence microscope, respectively. Results:Compared with the mice with CFT073 Δ tcpc infection, CFT073 wt-infected mice had significantly enlarged bladder and severe neutrophil infiltration and abundant TcpC in bladder tissues. The number of bacteria in the urine of CFT073 wt-infected mice was significantly greater than that of the CFT073 Δ tcpc group. PCR results showed that the bacteria in bladder or urine were CFT073 wt. The expression of TcpC at both mRNA and protein levels in macrophages increased significantly after CFT073 wt infection. Moreover, in CFT073 wt-infected macrophages, the expression of IκBα was promoted and the phosphorylation of p65 and the production of proinflammatory factors were suppressed. TcpC was instrumental in the survival and invasion of CFT073 wt in macrophages. Conclusions:TcpC expression increased significantly in mice with CFT073 wt-induced cystitis. TcpC inhibited the activation of NF-κB signaling pathway and the production of proinflammatory factors in macrophages to improve the survival rate of CFT073 wt, which was closely related to the pathogenesis and immune evasion of UPEC.