Effect and Mechanism of Mycobacterium tuberculosis Lipoprotein LpqH in NLRP3 Inflammasome Activation in Mouse Ana-1 Macrophage

Author:

Liu Liting1,Zhai Kaixin1,Chen Yue1,Chen Xiaowen1,Wang Guofu1,Wu Lixian1ORCID

Affiliation:

1. Department of Medical Microbiology and Immunology, Clinical Molecular Immunology Innovation Team, Dali University, Dali 671000, China

Abstract

The study is aimed at investigating the role and mechanism of LpqH of Mycobacterium tuberculosis in the activation of NLRP3 inflammasome in mouse Ana-1 macrophages. ExPASy-ProtParam, PHYRE2, ABCpred, and SYFPEITHI were used to predict and analyze the physicochemical properties, protein structure, and B cell/T cell-associated epitopes of LpqH protein. The recombinant LpqH protein was purified, and its immunoreactivity was analyzed with western blot. The LPS-treated mouse Ana-1 macrophages were incubated with purified LpqH protein directly. The expression of NLRP3, ASC, and caspase-1 protein was detected by western blot. The secretion of IL-1β was detected by ELISA, and LDH was detected by a kit. Cell death was detected by flow cytometry. LpqH consisted of 159 amino acids and was a hydrophobic protein with stable properties. Its secondary structure contained 47% random coils, 53% β-sheets, and 3% α-helix. The tertiary structure showed a relatively loose spatial conformation. Additionally, it had 8 B cell epitopes ( score > 0.8 ) and 10 CTL cell epitopes ( score 20 ). The recombinant LpqH, which had strong immunoreactivity, significantly increased the levels of NLRP3, ASC, and caspase-1 p20 ( P < 0.01 ) and promoted the secretion of IL-1β by the cells ( P < 0.01 ). In addition, high concentration of KCl significantly inhibited the effect of LpqH on mouse Ana-1 macrophages and reduced the expression of NLRP3, ASC, and caspase-1 p20 ( P < 0.01 ). However, there was no significant change in LDH ( P > 0.05 ). Meanwhile, LpqH protein did not cause additional cell death ( P > 0.05 ). LpqH protein has good immunogenicity and can activate the NLRP3 inflammasome through the potassium efflux pathway without causing cell death.

Funder

National Natural Science Foundation of China

Publisher

Hindawi Limited

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine

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