The synergistic pathogenic mechanism of Brucella melitensis M5-90 effector protein BspC in regulating host cell apoptosis, inflammation and oxidative stress

<b>Introduction.</b> Brucellosis is a zoonotic contact infectious disease caused by <i>Brucella</i>. Recently, researchers screened a series of <i>Brucella</i> effector proteins and identified the functions of most of them, which play important roles in the intracellular survival of <i>Brucella</i>.<b>Hypothesis.</b> We hypothesized that the BspC protein from <i>Brucella melitensis</i> M5-90 plays a significant role in the intracellular proliferation of <i>Brucella</i>.<b>Aim.</b> This study aimed to investigate the role of the <i>Brucella melitensis</i> M5-90 effector protein BspC in host cell processes, specifically its interaction with host proteins, and its effects on apoptosis, inflammation, oxidative stress, and global gene expression.<b>Methodology.</b> First, host proteins that interacted with BspC were identified from the cDNA library of a mouse macrophage cell line (RAW264.7) by yeast two-hybrid technology, and the key interacting proteins were confirmed by cotransformation. The pTT5-BspC recombinant plasmid was subsequently constructed and transfected into HEK293T cells. The expression of apoptosis-related proteins was assessed by Western blotting, the apoptosis rate was analysed by flow cytometry, the subcellular localization of proteins was observed by laser confocal microscopy and cytokines and oxidative stress indicators were assessed by ELISA and other kits. Furthermore, RNA-seq transcriptome sequencing was used to analyse the effects of BspC on the host gene expression profile.<b>Results.</b> Four host proteins (FDX1, DNAJA1, HSPA5 and PTPN2) that interacted with BspC were identified from the RAW264.7 cell cDNA library by yeast two-hybrid technology, and their interactions were confirmed by cotransformation. BspC protein expression in HEK293T cells significantly promoted cell apoptosis, as indicated by the upregulation of proapoptotic proteins (Bax, p53 and Caspase-3) and the downregulation of the antiapoptotic protein Bcl-2. Immunofluorescence staining revealed that BspC was localized in the cytoplasm and nucleus. In addition, BspC induced the secretion of proinflammatory cytokines (TNF-α, IL-1β and IL-6) and lactate dehydrogenase and reduced malondialdehyde levels by increasing the activities of SOD, CAT, GSH-PX and GSH, suggesting its regulation of the oxidative stress response. Transcriptome analysis revealed that BspC expression induced differential expression in 796 genes (209 upregulated and 587 downregulated), which were significantly enriched in the ECM-receptor interaction and MAPK and NF-κB signalling pathways.<b>Conclusion.</b> BspC may promote the immune escape and pathogenicity of <i>Brucella</i> by interfering with host cell apoptosis, the inflammatory response and the redox balance.