结核分枝杆菌Rv1043c蛋白酶的功能及在小鼠体内的致病性研究

doi:10.1016/j.jia.2023.06.025

摘要/Abstract

摘要：

结核病（Tuberculosis，TB）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，TB与HIV的混合感染以及多重耐药TB的出现，为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⁺和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-pH4.0和pH7.0-pH10.0的范围内二级结构稳定，在pH5.0和pH6.0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_Rv1043c和Msg_Rv1043c^S279A。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_Rv1043c^S279A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_pAIN、Msg_Rv1043c和Msg_Rv1043c^S279A感染C57BL/6小鼠，经病理学、组织病理学、肺细胞凋亡、菌落定殖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殖，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-1β、IL-6和TNF-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.

Key words: Mycobacterium tuberculosis , Mycobacterium smegmatis , serine protease , Rv1043c , Pathogenicity

TANG Yang-yang, CUI Ying-ying, JIANG Yan-yan, SHAO Ming-zhu, ZANG Xin-xin, DANG Guang-hui, LIU Si-guo. 结核分枝杆菌Rv1043c蛋白酶的功能及在小鼠体内的致病性研究[J]. Journal of Integrative Agriculture, 2023, 22(12): 3755-3768.

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

参考文献

Bah A, Sanicas M, Nigou J, Guilhot C, Astarie-Dequeker C, Vergne I. 2020. The lipid virulence factors of Mycobacterium tuberculosis exert multilayered control over autophagy-related pathways in infected human macrophages. Cells, 9, 666.

Behar S M, Martin C J, Booty M G, Nishimura T, Zhao X, Gan H X, Divangahi M, Remold H G. 2011. Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis. Mucosal Immunology, 4, 279–287.

Bernegger S, Vidmar R, Fonovic M, Posselt G, Turk B, Wessler S. 2021. Identification of desmoglein-2 as a novel target of Helicobacter pylori HtrA in epithelial cells. Cell Communication and Signaling, 19, 108.

Boehm M, Simson D, Escher U, Schmidt A M, Bereswill S, Tegtmeyer N, Backert S, Heimesaat M M. 2018. Function of serine protease HtrA in the lifecycle of the foodborne pathogen Campylobacter jejuni. European Journal of Microbiology and Immunology, 8, 70–77.

Burchacka E, Sienczyk M. 2018. The lord of the bacteria: The fellowship of the leader and other serine protease inhibitors. Current Pharmaceutical Design, 24, 4445–4465.

Chai Q, Wang L, Liu C H, Ge B. 2020. New insights into the evasion of host innate immunity by Mycobacterium tuberculosis. Cellular & Molecular Immunology, 17, 901–913.

Chen M, Gan H, Remold H G. 2006. A mechanism of virulence: virulent Mycobacterium tuberculosis strain H37Rv, but not attenuated H37Ra, causes significant mitochondrial inner membrane disruption in macrophages leading to necrosis. The Journal of Immunology, 176, 3707–3723.

Cui Y, Tang Y, Shao M, Zang X, Jiang Y, Cui Z, Dang G, Liu S. 2022. Mycobacterium tuberculosis protease Rv3090 is associated with late cell apoptosis and participates in organ injuries and mycobacterial dissemination in mice. Microbial Pathogenesis, 173, 105880.

Danelishvili L, Babrak L, Rose S J, Everman J, Bermudez L E. 2014. Mycobacterium tuberculosis alters the metalloprotease activity of the COP9 signalosome. mBio, 5, e01278-14.

Dang G, Chen L, Li Z, Deng X, Cui Y, Cao J, Yu S, Pang H, Liu S. 2015. Expression, purification and characterisation of secreted esterase Rv2525c from Mycobacterium tuberculosis. Applied Microbiology and Biotechnology, 176, 1–12.

Dang G, Cui Y, Wang L, Li T, Cui Z, Song N, Chen L, Pang H, Liu S. 2018. Extracellular sphingomyelinase Rv0888 of Mycobacterium tuberculosis contributes to pathological lung injury of Mycobacterium smegmatis in mice via inducing formation of neutrophil extracellular traps. Frontiers in Immunology, 9, 677.

Diedrich C R, O’hern J, Wilkinson R J. 2016. HIV-1 and the Mycobacterium tuberculosis granuloma: A systematic review and meta-analysis. Tuberculosis, 98, 62–76.

Goettig P, Brandstetter H, Magdolen V. 2019. Surface loops of trypsin-like serine proteases as determinants of function. Biochimie, 166, 52–76.

Gomez J E, Mckinney J D. 2004. M. tuberculosis persistence, latency, and drug tolerance. Tuberculosis, 84, 29–44.

Harish B S, Uppuluri K B. 2018. Microbial serine protease inhibitors and their therapeutic applications. International Journal of Biological Macromolecules, 107, 1373–1387.

Hedstrom L. 2002. Serine protease mechanism and specificity. Chemical Reviews, 102, 4501–4524.

Howell M, Dumitrescu D G, Blankenship L R, Herkert D, Hatzios S K. 2019. Functional characterization of a subtilisin-like serine protease from Vibrio cholerae. Journal of Biological Chemistry, 294, 9888–9900.

Jain R, Dey B, Khera A, Srivastav P, Gupta U D, Katoch V M, Ramanathan V D, Tyagi A K. 2011. Over-expression of superoxide dismutase obliterates the protective effect of BCG against tuberculosis by modulating innate and adaptive immune responses. Vaccine, 29, 8118–8125.

Ladel C H, Blum C, Dreher A, Reifenberg K, Kopf M, Kaufmann S H. 1997. Lethal tuberculosis in interleukin-6-deficient mutant mice. Infection and Immunity, 65, 4843–4849.

Lee J O, Kim J Y, Rhee D K, Pyo S. 2013. Streptococcus pneumoniae ClpP protease induces apoptosis via caspase-independent pathway in human neuroblastoma cells: Cytoplasmic relocalization of p53. Toxicon, 70, 142–152.

Li H, Dang G, Liu H, Wang Z, Cui Z, Song N, Chen L, Liu S. 2019. Characterization of a novel Mycobacterium tuberculosis serine protease (Rv3194c) activity and pathogenicity. Tuberculosis, 119, 101880.

Liu C H, Liu H, Ge B. 2017. Innate immunity in tuberculosis: Host defense vs pathogen evasion. Cellular & Molecular Immunology, 14, 963–975.

Liu M, Li W, Xiang X, Xie J. 2015. Mycobacterium tuberculosis effectors interfering host apoptosis signaling. Apoptosis, 20, 883–891.

Liu W, Spero D M. 2004. Cysteine protease cathepsin S as a key step in antigen presentation. Drug News & Perspectives, 17, 357–363.

Park H S, Back Y W, Jang I T, Lee K I, Son Y J, Choi H G, Dang T B, Kim H J. 2021. Mycobacterium tuberculosis Rv2145c promotes intracellular survival by STAT3 and IL-10 receptor signaling. Frontiers in Immunology, 12, 666293.

Pieters J. 2008. Mycobacterium tuberculosis and the macrophage: Maintaining a balance. Cell Host & Microbe, 3, 399–407.

Prokesova L, Potuznikova B, Potempa J, Zikan J, Radl J, Hachova L, Baran K, Porwit-Bobr Z, John C. 1992. Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus. Immunology Letters, 31, 259–265.

Qin S, Wang Z, Huang C, Huang P, Li D. 2022. Serine protease PRSS23 drives gastric cancer by enhancing tumor associated macrophage infiltration via FGF2. Frontiers in Immunology, 13, 955841.

Rahlwes K C, Dias B R S, Campos P C, Alvarez-Arguedas S, Shiloh M U. 2023. Pathogenicity and virulence of Mycobacterium tuberculosis. Virulence, 14, 2150449.

Rodriguez-Cabrera L, Trujillo-Bacallao D, Borras-Hidalgo O, Wright D J, Ayra-Pardo C. 2010. RNAi-mediated knockdown of a Spodoptera frugiperda trypsin-like serine-protease gene reduces susceptibility to a Bacillus thuringiensis Cry1Ca1 protoxin. Environmental Microbiology, 12, 2894–2903.

Samten B, Wang X, Barnes P F. 2009. Mycobacterium tuberculosis ESX-1 system-secreted protein ESAT-6 but not CFP10 inhibits human T-cell immune responses. Tuberculosis, 89(Suppl.1), S74–S80.

Sharafutdinov I, Esmaeili D S, Harrer A, Tegtmeyer N, Sticht H, Backert S. 2020. Campylobacter jejuni serine protease HtrA cleaves the tight junction component Claudin-8. Frontiers in Cellular and Infection Microbiology, 10, 590186.

Shin D M, Jeon B Y, Lee H M, Jin H S, Yuk J M, Song C H, Lee S H, Lee Z W, Cho S N, Kim J M, Friedman R L, Jo E K. 2010. Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling. PLoS Pathogens, 6, e1001230.

Skorko-Glonek J, Figaj D, Zarzecka U, Przepiora T, Renke J, Lipinska B. 2017. The extracellular bacterial HtrA proteins as potential therapeutic targets and vaccine candidates. Current Medicinal Chemistry, 24, 2174–2204.

Sodenkamp J, Waetzig G H, Scheller J, Seegert D, Grotzinger J, Rose-John S, Ehlers S, Holscher C. 2012. Therapeutic targeting of interleukin-6 trans-signaling does not affect the outcome of experimental tuberculosis. Immunobiology, 217, 996–1004.

Spoerry C, Karlsson J, Aschtgen M S, Loh E. 2021. Neisseria meningitidis IgA1-specific serine protease exhibits novel cleavage activity against IgG3. Virulence, 12, 389–403.

Sun Y, Wang Y, Liu W, Zhou J L, Zeng J, Wang X H, Jiang Y R, Li D H, Qin L. 2017. Upregulation of a trypsin-like serine protease gene in Antheraea pernyi (Lepidoptera: Saturniidae) strains exposed to different pathogens. Journal of Economic Entomology, 110, 941–948.

Tamgue O, Gcanga L, Ozturk M, Whitehead L, Pillay S, Jacobs R, Roy S, Schmeier S, Davids M, Medvedeva Y A, Dheda K, Suzuki H, Brombacher F, Guler R. 2019. Differential targeting of c-Maf, Bach-1, and Elmo-1 by microRNA-143 and microRNA-365 promotes the intracellular growth of Mycobacterium tuberculosis in alternatively IL-4/IL-13 activated macrophages. Frontiers in Immunology, 10, 421.

Velmurugan K, Chen B, Miller J L, Azogue S, Gurses S, Hsu T, Glickman M, Jacobs Jr W R, Porcelli S A, Briken V. 2007. Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells. PLoS Pathogens, 3, e110.

Wang J, Ge P, Qiang L, Tian F, Zhao D, Chai Q, Zhu M, Zhou R, Meng G, Iwakura Y, Gao G F, Liu C H. 2017. The mycobacterial phosphatase PtpA regulates the expression of host genes and promotes cell proliferation. Nature Communications, 8, 244.

Wen W, Qi Z, Wang J. 2020. The function and mechanism of Enterovirus 71 (EV71) 3C protease. Current Microbiology, 77, 1968–1975.

Xu G, Jia H, Li Y, Liu X, Li M, Wang Y. 2010. Hemolytic phospholipase Rv0183 of Mycobacterium tuberculosis induces inflammatory response and apoptosis in alveolar macrophage RAW264.7 cells. Canadian Journal of Microbiology, 56, 916–924.

Yan S, Wu G. 2021. Potential 3-chymotrypsin-like cysteine protease cleavage sites in the coronavirus polyproteins pp1a and pp1ab and their possible relevance to COVID-19 vaccine and drug development. FASEB Journal, 35, e21573.

Zhang L, Zhong Q, Bao L, Zhang Y, Gao L, Huang B, Zhang H D. 2009. Rv0901 from Mycobacterium tuberculosis, a possible novel virulent gene proved through the recombinant Mycobacterium smegmatis. Japanese Journal of Infectious Diseases, 62, 26–31.

Zhao Q, Li W, Chen T, He Y, Deng W, Luo H, Xie J. 2014. Mycobacterium tuberculosis serine protease Rv3668c can manipulate the host-pathogen interaction via Erk-NF-kappaB axis-mediated cytokine differential expression. Journal of Interferon & Cytokine Research, 34, 686–698.