Brucella is a facultative intracellular bacterium, dividing into smooth- and rough-type Brucella. Smooth-type Brucella can dissociate into rough mutants with cytotoxicity for macrophages during infection, which is critical for Brucella egress and dissemination. However, the mechanism of cytotoxicity infected by rough Brucella is incomplete. In this study, we verified that a rough-type Brucella (RB14 strain) was cytotoxic for macrophages dependent on Type IV secretion system (T4SS). Two specific T4SS VirB4 and VirB11 mutants were constructed, which affect the secretion of T4SS effectors, but not the expression of T4SS components. Cytotoxicity analysis showed that RB14- induced macrophages death depends on T4SS secretion activity. In a further study, 15 reported T4SS effectors were evaluated in inducing macrophage death using over-expression and transfection methods, the results showed that 15 recombinant strains with over-expression of respective effector were not cytotoxicity. In addition, 10 effectors transfected individually, or co-transfected with five effectors barely induced macrophage death, suggesting that all 15 effectors were not associated with macrophage death. Besides, we also evaluated endoplasmic reticulum (ER) stress, Txnip- or Caspase-2 roles in RB14-induced macrophages death. The results showed that inhibition of ER stress, Caspase or Caspase-2 activation was not associated with RB14-infected macrophages death. The casp2 and txnip knockout cells also showed death when infected by the RB14 strain. In all, the RB14-induced macrophage death depends on the secretion activity of T4SS, but not on ER stress, Txnip- or Caspase-2 signal pathway. This study provides a deep insight for rough Brucella-induced macrophage death, which favors for elucidating Brucella infection lifecycle.

中文翻译：

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粗略的布鲁氏菌突变体诱导的巨噬细胞死亡取决于T4SS的分泌活性，而不取决于细胞的Txnip和Caspase-2介导的信号通路。

布鲁氏菌是兼性的细胞内细菌，分为平滑型和粗糙型布鲁氏菌。平滑型布鲁氏菌可在感染过程中解离为对巨噬细胞具有细胞毒性的粗糙突变体，这对于布鲁氏菌的外出和传播至关重要。但是，粗布鲁氏菌感染的细胞毒性机制并不完整。在这项研究中，我们验证了粗糙型布鲁氏菌（RB14株）对依赖于IV型分泌系统（T4SS）的巨噬细胞具有细胞毒性。构建了两个特定的T4SS VirB4和VirB11突变体，它们影响T4SS效应子的分泌，但不影响T4SS成分的表达。细胞毒性分析表明，RB14诱导的巨噬细胞死亡取决于T4SS的分泌活性。在进一步的研究中 使用过量表达和转染方法评估了15种已报道的T4SS效应子在诱导巨噬细胞死亡中的作用，结果表明15种具有各自表达效应的重组菌株没有细胞毒性。另外，单独转染或与五个效应子共转染的10个效应子几乎不诱导巨噬细胞死亡，这表明所有15个效应子均与巨噬细胞死亡无关。此外，我们还评估了内质网应激（ER），Txnip-或Caspase-2在RB14诱导的巨噬细胞死亡中的作用。结果表明，抑制内质网应激，Caspase或Caspase-2活化与RB14感染的巨噬细胞死亡无关。当被RB14株感染时，casp2和txnip基因敲除细胞也显示死亡。在所有，RB14诱导的巨噬细胞死亡取决于T4SS的分泌活性，而不取决于内质网应激，Txnip或Caspase-2信号通路。这项研究为布鲁氏菌引起的巨噬细胞死亡提供了深刻的见解，有助于阐明布鲁氏菌感染的生命周期。