Abstract
Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular mechanisms associated with Brucella rough mutant-induced macrophage cytotoxicity.
Highlights
Brucella is a Gram-negative facultative intracellular bacterial species that causes zoonotic brucellosis, characterized by reproductive disease in domestic animals and chronic debilitating disease in humans (Boschiroli et al, 2001; Franco et al, 2007; Whatmore, 2009)
We demonstrated that VjbR upregulation in the Brucella rough mutant rfbE enhances T4SS expression and secretion, both of which contribute to the death of infected macrophages via activation of the inositol-requiring enzyme 1α (IRE1α) pathway of endoplasmic reticulum (ER) stress
Morphology of the RAW264.7 cells infected with S2308, rfbE, rfbE(pBBR-rfbE), and rfbE virB were observed via light microscopy
Summary
Brucella is a Gram-negative facultative intracellular bacterial species that causes zoonotic brucellosis, characterized by reproductive disease in domestic animals and chronic debilitating disease in humans (Boschiroli et al, 2001; Franco et al, 2007; Whatmore, 2009). Brucella LPS is composed of lipid A, a core oligosaccharide, and the O-antigen It is characterized by low stimulatory activity and toxicity to cells, and mediates lower superoxide and lysozyme production in infected cells (Goldstein et al, 1992; Rasool et al, 1992). The rough mutant VTRS1 of B. suis induced proinflammatory, caspase-2- and nuclear factor kappa B (NFκB)- mediated macrophage cell death (Chen et al, 2011). Bronner and colleagues subsequently reported that the rough mutant of B. abortus RB51 induces a hybrid cell death, mediated by caspase-2 activation, with features of apoptosis and pyroptosis (Bronner et al, 2013). The molecular mechanism underlying Brucella rough mutant modulation of ER stress to induce macrophage death remains unclear
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