利用Tet-On-3G系统构建可诱导表达ESAT-6的THP-1细胞系,研究结核分枝杆菌毒力蛋白ESAT-6在宿主细胞内的生物学功能。首先,利用聚合酶链反应(PCR)扩增ESAT-6片段和红色荧光蛋白mCherry片段,并与pLVX-TRE3G载体连接,构建pTRE3G-mCherry-ES

为探索牛分枝杆菌通过RIPK1来调节细胞凋亡的作用机制,本试验利用分子克隆技术构建诱饵质粒pGBKT7-RIPK1,并通过PEG/LiAc转化法将该质粒转入酵母菌中,经Western blot验证RIPK1在酵母菌中的表达情况后,使用该重组酵母菌筛选牛分枝杆菌基

Pathogenic mycobacteria, such asMycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium marinum,can trigger NLRP3 inflammasome activation leading to maturation and secretion of interleukin 1 beta (IL-1 beta). However, the mycobacterial factors involved in the activation of NLRP3 inflammasome are not fully understood. Here, we identified that the PPE family protein PPE13 was responsible for the induction of IL-1 beta secretion in a NLRP3 inflammasome-dependent manner. We found that the recombinantMycobacterium smegmatisexpressing PPE13 activates NLRP3 inflammasome, thereby inducing caspase-1 cleavage and IL-1 beta secretion in J774A.1, BMDMs, and THP-1 macrophages. To examine whether this inflammasome activation was triggered by PPE13 rather than components ofM. smegmatis, PPE13 was introduced into the aforementioned macrophages by lentivirus as a delivery vector. Similarly, this led to the activation of NLRP3 inflammasome, indicating that PPE13 is a direct activator of NLRP3 cascade. We further demonstrated that the NLRP3 complex activated the inflammasome cascade, and the assembly of this complex was facilitated by PPE13 through interacting with the LRR and NATCH domains of NLRP3. Finally, we found that all PPE13 proteins isolated fromM. tuberculosis,M. bovis,andM. marinumcan activate NLRP3 inflammasome through binding to NLRP3, which requires C-terminal repetitive MPTR domain of PPE13. Thus, we, for the first time, revealed that PPE13 triggers the inflammasome-response by interacting with the MPTR domain of PPE13 and the LRR and NATCH domains of NLRP3. These findings provide a novel perspective on the function of PPE proteins in the immune system during mycobacteria invasion.

Ginsenoside Rg3 is one of the main constituents of Panax ginseng. Compelling evidence has demonstrated that ginsenoside Rg3 is capable of inhibiting inflammation. However, the mechanism mediating its anti-inflammatory effects remain unclear. Here we show that ginsenoside Rg3 blocks IL-1 beta secretion and caspase-1 activation through inhibiting LPS priming and the NLRP3 inflammasome activation in human and mouse macrophages. Rg3 specifically inhibits activation of NLRP3 but not the NLRC4 or AIM2 inflammasomes. In addition, Rg3 has no effect on upstream regulation of NLRP3 inflammasome, such as K+ efflux, ROS production, or mitochondrial membrane potential. Mechanistically, Rg3 abrogates NEK7-NLRP3 interaction, and subsequently inhibits NLRP3-ASC interaction, ASC oligomerization, and speckle formation. More importantly, Rg3 can reduce IL-1 beta secretion induced by LPS in mice and protect mice from lethal endotoxic shock. Thus, our findings reveal an anti-inflammatory mechanism for Rg3 and suggest its potential use in NLRP3-driven diseases.

We developed a multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 6 clinically relevant viral pathogens causing porcine reproductive failure, that is porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV) and porcine parvovirus (PPV). The limits of detection for the assay varied among the 6 target organisms from 1 to 8 copies per MLPA assay. The MLPA assay was evaluated with 346 heparinized porcine umbilical cord blood specimens, and the results of the assay were compared to those of real-time PCR. The MLPA assay showed specificities and sensitivities of 99.2% and 100%, respectively, for PRRSV; 100% and 100%, respectively, for CSFV, PCV2, PRV and PPV. No sample was found to be positive for JEV by either the MLPA assay or the real-time PCR. In conclusion, the MLPA assay has comparable clinical sensitivity to that of real-time PCR assay and provides a useful tool for fast screening porcine reproductive failure-associated viruses.

The NLRP3 inflammasome is a multimeric protein complex that initiates an inflammatory form of cell death and triggers the release of proinflammatory cytokines IL-1 beta and IL-18. The NLRP3 inflammasome has been implicated in a wide range of diseases, including Alzheimer's disease, Prion diseases, type 2 diabetes, and some infectious diseases. It has been found that a variety of stimuli including danger-associated molecular patterns (DAMPs, such as silica and uric acid crystals) and pathogen-associated molecular patterns (PAMPs) can activate NLRP3 inflammasome, but the specific regulatory mechanisms of NLRP3 inflammasome activation remain unclear. Understanding the mechanisms of NLRP3 activation will enable the development of its specific inhibitors to treat NLRP3-related diseases. In this review, we summarize current understanding of the regulatory mechanisms of NLRP3 inflammasome activation as well as inhibitors that specifically and directly target NLRP3.

Thiol-disulfide glutaredoxin systems of bacterial cytoplasm favor reducing conditions for the correct disulfide bonding of functional proteins, and therefore were employed by bacteria to defend against oxidative stress. Listeria monocytogenes has been shown to encode a putative glutaredoxin, Grx (encoded by lmo2344), while the underlying roles remain unknown. Here we suggest an unexpected role of L. monocytogenes Grx in oxidative tolerance and intracellular infection. The recombinant Grx was able to efficiently catalyze the thiol-disulfide oxidoreduction of insulin in the presence of DTT as an election donor. Unexpectedly, the deletion of grx resulted in a remarkably increased tolerance and survival ability of this bacteria when exposed to various oxidizing agents, including diamide, and copper and cadmium ions. Furthermore, loss of grx significantly promoted bacterial invasion and proliferation in human epithelial Caco-2 cells and murine macrophages, as well as a notably increasing invasion but not cell-to-cell spread in the murine fibroblasts L929 cells. More importantly, L. monocytogenes lacking the glutaredoxin exhibited more efficient proliferation and recovery in the spleens and livers of the infected mice, and hence became more virulent by upregulating the virulence factors, InlA and InlB. In summary, we here for the first time demonstrated that L. monocytogenes glutaredoxin plays a counterintuitive role in bacterial oxidative resistance and intracellular infection, which is the first report to provide valuable evidence for the role of glutaredoxins in bacterial infection, and more importantly suggests a favorable model to illustrate the functional diversity of bacterial Grx systems during environmental adaption and host infection.

The NLRP3 inflammasome is a multimeric protein complex that initiates an inflammatory form of cell death and triggers the release of proinflammatory cytokines IL-1 beta and IL-18. The NLRP3 inflammasome has been implicated in a wide range of diseases, including Alzheimer's disease, Prion diseases, type 2 diabetes, and some infectious diseases. It has been found that a variety of stimuli including danger-associated molecular patterns (DAMPs, such as silica and uric acid crystals) and pathogen-associated molecular patterns (PAMPs) can activate NLRP3 inflammasome, but the specific regulatory mechanisms of NLRP3 inflammasome activation remain unclear. Understanding the mechanisms of NLRP3 activation will enable the development of its specific inhibitors to treat NLRP3-related diseases. In this review, we summarize current understanding of the regulatory mechanisms of NLRP3 inflammasome activation as well as inhibitors that specifically and directly target NLRP3.

为研究牛分枝杆菌抑制肿瘤坏死因子介导的细胞凋亡来逃避宿主免疫反应的机制,本试验采用酵母双杂交系统在牛分枝杆菌中筛选可与肿瘤坏死因子受体1相关死亡域蛋白（TRADD）相互作用的蛋白。通过限制性内切酶Sau3AⅠ部分消化牛分枝杆菌基因组,回收片段随机插入pGADT7载体中,转化大肠杆菌DH5α感受态细胞,构建牛分枝杆菌基因组文库。PCR扩增人tradd基因,将扩增产物克隆于pGBKT7载体上,构建重组诱饵质粒pGBKT7-tradd,转化酵母菌Y2HGold。用诱饵质粒pGBKT7-tradd对牛分枝杆菌基因组文库进行筛选,以获得与TRADD互作的阳性候选克隆;提取阳性候选克隆中的质粒,经测序和同源性比对分析,获得与TRADD互作的牛分枝杆菌蛋白的生物学信息。结果显示,构建的文库滴度为2×10~6 CFU,平均插入片段在1.5kb左右,文库重组率＞95%。经Western blotting验证,诱饵质粒pGBKT7-tradd可在酵母菌中表达诱饵蛋白TRADD,且TRADD的表达对酵母菌无毒性,不会在酵母菌中自激活。应用酵母双杂交系统初步筛选出20个与TRADD互作的阳性克隆,经复筛、测序和BLAST对比,最终发现7个基因序列。本研究应用酵母双杂交技术成功筛选到7个与TRADD互作的牛分枝杆菌蛋白,为进一步研究牛分枝杆菌对细胞凋亡的抑制机制提供线索。

牛分枝杆菌是重要的人畜共患病原菌，可通过抑制TNF介导的细胞凋亡来促进细菌的增殖和扩散，逃避树突状细胞等抗原递呈细胞对分枝杆菌抗原的递呈，延缓机体获得性免疫反应的发生。与之相反，经传代致弱的牛分枝杆菌BCG则可促进细胞凋亡的发生，激活更强烈的获得性免疫反应。然而，牛分枝杆菌强毒株抑制细胞凋亡的机制还不清楚。TNF可以使其受体TNFR三聚化并招募死亡结构域相关蛋白TRADD、FADD和RIP1形成凋亡诱导复合体，从而导致细胞凋亡的发生。那么，牛分枝杆菌中哪些蛋白参与了凋亡诱导复合体形成的调控呢?本项目首先通过酵母双杂交技术，使用TNF信号通路中三个重要的效应蛋白TRADD、FADD和RIP1筛选了牛分枝杆菌基因组文库，经回补验证、测序分析、Blast比对以及CO-IP验证，最终发现了与TRADD和FADD均互相作用的牛分枝杆菌蛋白PPE13,以及与RIP1互作的PE16、sseB、Mb0869c和Mb0383c。再次，探讨了PPE13牛分枝杆菌PPE13蛋白对TNF介导的信号通路的影响。在TNF介导的NF-κB活化以及细胞凋亡模型中,PPE13不仅抑制TNF引起的NF-κB下游因子IκB的降解和磷酸化的形成，还抑制由TNF和CHX引发的细胞凋亡的发生。另外,PPE13也抑制耻垢分枝杆菌引起的细胞凋亡，但同时促进细胞焦亡的发生。最后，构建了TRADD过表达引起的细胞死亡的模型，并通过该模型确定了与RIP1互作蛋白中PE16、sseB和Mb0383c可抑制细胞死亡的发生，其中Mb0383c的抑制作用最强。总之，通过本项目的研究，我们发现了牛分枝杆菌中参与抑制细胞凋亡的相关蛋白，为进一步探索牛分枝杆菌感染机制提供了理论基础。