A rough Brucella mutant induced macrophage death depends on secretion activity of T4SS, but not on cellular Txnip- and Caspase-2-mediated signaling pathway

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.