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Journal of Integrative Agriculture  2023, Vol. 22 Issue (12): 3755-3768    DOI: 10.1016/j.jia.2023.06.025
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
Characteristics of Mycobacterium tuberculosis serine protease Rv1043c in enzymology and pathogenicity in mice
TANG Yang-yang*, CUI Ying-ying*, JIANG Yan-yan, SHAO Ming-zhu, ZANG Xin-xin, DANG Guang-hui#, LIU Si-guo#

State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, P.R.China

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摘要  

结核病(TuberculosisTB)是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的一种严重危害人类健康并对畜牧业造成巨大威胁的人畜共患传染病。近年来,TB在全球范围内流行更为广泛,根据2022年世界卫生组织(WHO)的报告,2022年共有987万人患TB,其中死亡128万人。同时TBHIV的混合感染以及多重耐药TB的出现TB的防控带来了新的挑战因此,解析Mtb的致病机制迫在眉睫。

丝氨酸蛋白酶在细菌入侵宿主以及免疫调节过程中发挥重要作用,我们的研究发现Rv1043c蛋白是Mtb细胞壁相关的丝氨酸蛋白酶,1-18位氨基酸为其信号肽序列,在致病性分枝杆菌中高度保守。我们以Rv1043c蛋白为研究对象,开展了该蛋白的酶学特性及其在致病性中的研究。

首先通过原核表达和亲和层析纯化获得Rv1043c蛋白,酶活性测定结果显示Rv1043c能够切割Casein底物,其最适温度为45 ℃、最适pH值为9.0,且Ca2+ Mg2+能够促进其活性的发挥,279位丝氨酸(S)为蛋白酶的关键活性位点,表明Rv1043c蛋白酶为丝氨酸蛋白酶圆二色分析结果显示,Rv1043c蛋白酶在温度10 ℃-70 ℃pH3.0-pH4.0pH7.0-pH10.0的范围内二级结构稳定,在pH5.0pH6.0条件下Rv1043c蛋白二级结构稳定性发生改变。

为了研究Rv1043c在分枝杆菌感染中的作用,我们构建了重组耻垢分枝杆菌Msg_pAIN、Msg_Rv1043cMsg_Rv1043cS279A。亚细胞定位和免疫电镜分析显示,Rv1043c蛋白定位于Mtb的表面;重组菌Msg_pAIN、Msg_Rv1043c和Msg_Rv1043cS279A感染小鼠腹腔巨噬细胞,经细胞毒性和凋亡分析显示,Rv1043c丝氨酸蛋白酶活性能够明显促进小鼠腹腔巨噬细胞的LDH释放;重组菌Msg_pAINMsg_Rv1043cMsg_Rv1043cS279A感染C57BL/6小鼠,经病理学、组织病理学、肺细胞凋亡、菌落定和炎性细胞因子分析显示,Rv1043c丝氨酸蛋白酶活性促进分枝杆菌在小鼠肺脏中的定,诱导肺细胞凋亡和肺的病理损伤,促进小鼠炎性细胞因子IL-1βIL-6TNF-α的释放。

以上研究结果表明Rv1043c丝氨酸蛋白酶在分枝杆菌致病过程中发挥重要作用本研究为进一步解析Mtb的致病机制提供理论基础。



Abstract  

The serine proteases of Mycobacteria tuberculosis (Mtb) are important contributors to the process of bacterial invasion and its pathogenesis.  In the present study, we systematically characterized the role of the Rv1043c protein in Mycobacterium infection by purifying the Rv1043c protein in Escherichia coli and constructing a Mycobacterium smegmatis (Msg) strain overexpressing Rv1043c (Msg_Rv1043c).  We found that Rv1043c had serine protease activity and localized to the surface of Mtb.  We determined that the optimal pH and temperature for the Rv1043c serine protease were 9.0 and 45°C, respectively.  Moreover, the serine protease activity of Rv1043c was enhanced by divalent metal ions of Ca2+ and Mg2+.  Site-directed mutagenesis studies demonstrated that the serine 279 residue in Rv1043c plays a catalytic role.  Additionally, mouse model studies confirmed that Rv1043c significantly enhanced the survival of Msg in vivo, induced pulmonary injury and lung cell apoptosis, and promoted the release of pro-inflammatory cytokines interleukin-1β and interleukin-6 in mice.  This study presents novel insights into the relationship between mycobacterial serine protease and the pathogenesis of the disease.

Keywords:  Mycobacterium tuberculosis        Mycobacterium smegmatis        serine protease        Rv1043c        Pathogenicity  
Received: 19 January 2023   Accepted: 31 May 2023
Fund: This research was supported by the National Key Research and Development Program of China (2021YFD1800403) and the National Natural Science Foundation of China (32273005 and 32002256).
About author:  TANG Yang-yang, E-mail: 994527709@qq.com; CUI Ying-ying, E-mail: cuiyingying1104@163.com; #Correspondence DANG Guang-hui, E-mail: dangguanghui@caas.cn; LIU Si-guo, Tel: +86-451-51051731, E-mail: liusiguo@caas.cn * These authors contributed equally to this study. * co-first authors.

Cite this article: 

TANG Yang-yang, CUI Ying-ying, JIANG Yan-yan, SHAO Ming-zhu, ZANG Xin-xin, DANG Guang-hui, LIU Si-guo. 2023. Characteristics of Mycobacterium tuberculosis serine protease Rv1043c in enzymology and pathogenicity in mice. Journal of Integrative Agriculture, 22(12): 3755-3768.

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