Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages.
Caspase-1-dependent and -independent cell death pathways in Burkholderia pseudomallei infection of macrophages.
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DOI：
10.1371/journal.ppat.1003986
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发表时间：
2014-03
期刊：
影响因子：
6.7
通讯作者：
Steinmetz I
中科院分区：
文献类型：
--
作者：
Bast A;Krause K;Schmidt IH;Pudla M;Brakopp S;Hopf V;Breitbach K;Steinmetz I
假假伯克氏菌胞质病原体和类鼻疽病病原体已被证明通过nod样受体NLRP3调节IL-1β和IL-18的产生，并通过NLRC4调节焦亡。到目前为止，这些受体的下游信号通路和假芽孢杆菌感染期间诱导的其他细胞死亡机制尚未得到详细解决。此外，假芽孢杆菌因子在炎性小体激活中的作用仍不明确。在本研究中，我们发现在巨噬细胞感染的早期阶段，caspase-1的加工和凋亡完全依赖于NLRC4，而不依赖于NLRP3，而在较晚的时间点，caspase-1的激活和细胞死亡则不依赖于NLRC4。在早期阶段，我们发现了NLRC4和caspase-1下游涉及caspase- 9、-7和PARP的激活途径。对caspase-1/11缺陷感染的巨噬细胞的分析显示，caspase-1/11缺陷感染的巨噬细胞强烈诱导凋亡，这依赖于凋亡引发剂和效应caspase的激活。在感染假芽孢杆菌鞭毛蛋白FliC或T3SS3 BsaU突变体后，巨噬细胞中caspase-1的早期激活途径明显减少或完全消失。使用转染野生型和突变T3SS3效应蛋白hope的细胞进行的研究也表明，该蛋白在caspase-1加工中也起作用。与野生型相比，T3SS3内棒蛋白BsaK突变体未能激活caspase-1，在巨噬细胞感染早期（而在晚期）显示出更高的细胞内计数，细胞死亡和IL-1β分泌减少。携带BsaK突变体的BALB/c小鼠鼻内感染显示死亡率显著降低，器官细菌负荷降低，支气管肺泡灌洗液中IL-1β、髓过氧化物酶和中性粒细胞水平降低。总之，我们的研究结果表明，功能性T3SS3在nlrc4介导的早期caspase-1激活和焦亡中发挥了重要作用，并在假芽孢杆菌感染的发病机制中参与了晚期caspase-1依赖和独立的细胞死亡机制。炎性小体的激活对宿主防御细菌感染很重要。许多革兰氏阴性病原体利用分泌系统将细菌蛋白（如鞭毛蛋白）或分泌机制本身的结构成分注入宿主细胞质，导致caspase-1激活和热腐细胞死亡。然而，对于触发caspase-1激活的假芽孢杆菌因子以及caspase-1的下游信号通路和效应机制知之甚少。在这里，我们发现假芽孢杆菌T3SS3内杆蛋白BsaK是原代巨噬细胞中caspase-1依赖性细胞死亡和IL-1β分泌的早期激活因子，也是小鼠类melidosis的毒力因子。我们可以证明，在巨噬细胞感染后，caspase-7在NLRC4/caspase-1炎性体下游被激活，并需要caspase-9加工。尽管caspase-7对DNA损伤传感器PARP的裂解至关重要，但它既不促进细胞因子的产生，也不通过促进巨噬细胞早期死亡来限制假芽孢杆菌的生长。除了在野生型巨噬细胞中快速诱导NLRC4/caspase-1依赖的焦亡外，我们还观察到在缺乏caspase-1/11的巨噬细胞中，经典凋亡的激活延迟。因此，启动不同的细胞死亡途径似乎是限制细胞内假芽孢杆菌感染的有效策略。
The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection. Inflammasome activation is important for host defence against bacterial infection. Many gram-negative pathogens use secretion systems to inject bacterial proteins such as flagellin or structural components of the secretion machinery itself into the host cytosol leading to caspase-1 activation and pyroptotic cell death. However, little is known about the B. pseudomallei factors that trigger caspase-1 activation as well as downstream signalling pathways and effector mechanisms of caspase-1. Here, we identified the B. pseudomallei T3SS3 inner rod protein BsaK as an early activator of caspase-1-dependent cell death and IL-1β secretion in primary macrophages and as a virulence factor in murine melioidosis. We could show that upon infection of macrophages, caspase-7 is activated downstream of the NLRC4/caspase-1 inflammasome and requires caspase-9 processing. Although caspase-7 was essential for cleavage of the DNA damage sensor PARP during pyroptosis, it did neither contribute to cytokine production nor B. pseudomallei growth restriction by promoting early macrophage death. In addition to a rapid NLRC4/caspase-1- dependent induction of pyroptosis in wild-type macrophages, we observed a delayed activation of classical apoptosis in macrophages lacking caspase-1/11. Thus, initiation of different cell death pathways seems to be an effective strategy to limit intracellular B. pseudomallei infection.
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影响因子：
5.8
作者：
Gereke M;Gröbe L;Prettin S;Kasper M;Deppenmeier S;Gruber AD;Enelow RI;Buer J;Bruder D
通讯作者：
Bruder D
影响因子：
3.2
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影响因子：
3.1
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16
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通讯作者：
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影响因子：
5.4
作者：
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通讯作者：
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