Molecular Mechanism Of Brucella Rough Mutant Induces Macrophage Death Via Activating T4SS

Brucella is a facultative intracellular pathogen that causes a worldwide-distributed zoonosis known as brucellosis,causing abortion in animals and undulant fever in human.Brucella virulence mostly relies on its ability to invade into and replicate within professional and non-professional phagocytic cells.Brucella express a series of virulence factors to ensure its survival and replication after invading host cells,and lipopolysaccharide?LPS?and Type IV secretion system?T4SS?are two major Brucella virulence factors.Based on LPS integrity,Brucella can be divided into smooth and rough phenotypes.Rough mutants induce infected macrophage death,which is T4 SS dependent.However,the molecular mechanism remains unclear.In this study,we used the luciferase reporter gene,Western blotting,qPCR to detect the molecular mechanism,and the proteome of secretory proteins from Brucella abortus was analyzed by Label-free method to detect the crucial secretory proteins.In this study,to identify the role of T4 SS on ?rfbE mutant-induced cytotoxicity for macrophages,the death of S2308,?rfbE and ?rfbE?virB123-infected macrophages was analyzed by FITC-Annexin V and propidium iodide staining and lactate dehydrogenase?LDH?release.The results confirmed that rough mutant ?rfbE induced macrophages death is T4 SS dependent.To detect the function of T4 SS in the macrophages death induced by ?rfbE,T4 SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparative investigated by constructing firefly luciferase fused T4 SS effector BPE123 and VceC.The results showed that the level of T4 SS secretion in rough mutant ?rfbE was higher than smooth strain S2308.To confirm that the enhanced T4 SS secretion is associated with enhanced T4 SS expression in Brucella rough mutant,qPCR and western blotting were used to analyze the T4 SS expression at transcriptional and translational levels respectively.The results showed that T4 SS expression and secretion were enhanced significantly in Brucella rough mutant compared to its smooth wild-type strain under vegetative,nutrition deprivation and acid conditions.In addition,using luciferase reporter assay we found that the activity of T4 SS virB operon promoter was obviously increased in Brucella rough mutant under vegetative,acid and intracellular growth conditions.Taken together,T4 SS expression and secretion were enhanced significantly in the Brucella rough mutant compared to its smooth wild-type strain.To investigate whether T4 SS over-expression in the rough mutant is associated with transcriptional regulators directly binding to the virB operon,we detect the expression of transcriptional regulators using qPCR,showing that expression of the vjbR was significantly upregulated,and of the mdrA and blxR were obviously down-regulated in the ?rfbE mutant,compared to the S2308 strain.To determine whether macrophage death caused by the ?rfbE mutant is associated with VjbR,MdrA and BlxR,we constructed ?rfbE?vjbR,?rfbE?pMdrA?and ?rfbE?pBlxR?strains to infect macrophages,showing that VjbR regulation plays a key role in Brucella rough mutant induced macrophage death and BlxR regulation also plays a part role in this process.To determine whether vjbR and blxR are essential to T4SS-upregulated expression in the ?rfbE mutant,the virB4 expression of ?rfbE?vjbR and ?rfbE?pBlxR?strains were detected by qPCR,suggesting that T4SS-upregulated expression in the ?rfbE is associated with regulatory protein VjbR and BlxR.To investigate the endoplasmic reticulum?ER?stress in Brucella rough mutant which induced by T4 SS effector proteins,the activation of p-IRE1? was analyzed by Western blotting,indicating that Brucella rough mutant induced stronger ER stress.To determine whether p-IRE1? is involved in macrophage death caused by the ?rfbE mutant,we evaluate the death of 4?8c-treated macrophages infected with S2308 and the ?rfbE mutant,indicating that macrophage death caused by the ?rfbE mutant is IRE1? dependent.Taken together,up-regulation of T4 SS promoted by VjbR and BlxR in rough mutant ?rfbE contributes to macrophage death macrophage death via strongly activating IRE1? pathway of ER stress.The proteome of secretory proteins from S2308,?rfbE and ?rfbE?virB123 was analyzed by liquid chromatography/tandem mass spectrometry to detect the crucial secretory proteins which is involved in macrophages death induced by Brucella rough mutant.To prepare protein samples,S2308,?rfbE and ?rfbE?virB123 were grown to log phase and exposed to RPMI-1640 for 8 h,indicating that the level of secretory proteins in rough mutant ?rfbE was higher than smooth strain S2308.After spectrometry analysis,1131 unique proteins were finally identified.The results of GO enrichment analysis demonstrated that differential secretory proteins are mainly involved in cellular process and metabolic process,distributed in the cell and membrane,and possessed molecular function of catalytic activity and binding.KEGG pathway analysis for the differential secretory proteins from ?rfbE and ?rfbE?virB123 showed that secretory proteins were associated with the Ribosome,NOD-like receptor signaling pathway,Two-component system and Bacterial secretion system.To determine whether macrophage death caused by infection with the ?rfbE mutant is associated with differential secretory proteins,we analyze the macrophage cytotoxicity induced by smooth or rough Brucella which delete or overexpress the differential secretory proteins.The result indicated that OmpW family protein?BAB1₁579?and uncharacterized protein?BAB1₁185?were associated with Brucella rough mutant-induced macrophage cytotoxicity.In addition,we found that Brucella rough mutant-induced macrophage cytotoxicity is caused by multiple T4 SS effectors,not T4 SS component proteins.Taken together,this study provides evidence that compared to Brucella smooth wild-type strain,VjbR up-regulation in Brucella rough mutant increased the transcription of virB operon,resulting in T4 SS gene over-expression accompanied by effector proteins over-secretion,such as VceC,OmpW family protein?BAB1₁579?and uncharacterized protein?BAB1₁185?,which causes infected macrophage death via activating IRE1? pathway of ER stress.This study provides novel insights into molecular mechanism of Brucella rough mutant induced macrophage cytotoxicity.