Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

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.

Relationship Between Quorum Sensing and Secretion Systems

“…Several T4SS ( virB operon) are controlled by VjbR protein which is a LuxR-type quorum-sensing regulator in B. abortus (Arocena et al, 2010; Li et al, 2017). Moreover, in the Roseobacter group, the conjugation of plasmids, which encode T4SS, is QS-controlled and the QS system may detect a broad range of long-chain AHLs at the cell surface (Patzelt et al, 2013, 2016).…”

“…In Brucella abortus , there is a clear relationship between the QS network and T4SS. For the virB operon, which encodes the T4SS regulated by VjbR, a LuxR-type QS is responsible for the virulence characteristics of B. abortus (Li et al, 2017). The virB operon is responsible for establishing the replicative niche of the bacterium once it enters the host cell.…”

“…The T4SS in B. abortus , as in other bacteria, translocates effector proteins into the host cell to avoid the immune defense mechanisms, making it one of the two main virulence factors for Brucella. Arocena et al (2010) described the binding site of VjbR to the virB operon (Li et al, 2017). Otherwise, the conjugation process between two members of the Roseobacter group mediated by T4SS, encoded in RepABC-type plasmids, is controlled by the QS network.…”

Quorum-Sensing Regulation of Antimicrobial Resistance in Bacteria

“…The QS system is directly related to T4SS in Brucella abortus. For the virB operon encoding T4SS regulated by VjbR, LuxR-type QS is responsible for the virulence characteristics of Brucella abortus [100].…”

The putative amino acid ABC transporter substrate-binding protein AapJ2 is necessary for Brucella virulence at the early stage of infection in a mouse model

“…RAW 264.7 macrophages were used to assess the ability of the Δ27735 mutant to survive intracellularly. The experiment was performed as previously reported [ 14 , 15 ]. Briefly, cells were seeded in 24-well plates and grown in Dulbecco’s Modified Eagle Medium (DMEM) (Hyclone™; GE Healthcare, Chalfont St. Giles, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco ® ; Invitrogen Corporation, Carlsbad, CA, USA) at 37 °C under an atmosphere of 5% CO 2 for 24 h. The cell monolayer was washed twice with DMEM and infected with the WT strain or the Δ27735 mutant at a multiplicity of infection (MOI) of 200.…”