关键词: 巨噬细胞, 鸟苷酸结合蛋白2, 金黄色葡萄球菌, MAPK信号通路, 细胞凋亡

Abstract:

【Background】Staphylococcus aureus is a common gram-positive pathogenic bacterium capable of causing various infectious diseases and toxin-mediated illnesses. As central effectors of innate immunity, macrophage play a critical role in eliminating S. aureus. However, S. aureus can evade immune clearance by inducing macrophage apoptosis. Guanylate-binding protein 2 (GBP2), an interferon-inducible GTPase, participates in host immune regulation against intracellular pathogens, but its specific functions and regulatory mechanisms in S. aureus-induced macrophage apoptosis remain unclear.【Objective】This study aimed to investigate the regulatory role of GBP2 and its underlying molecular mechanisms in macrophage apoptosis during S. aureus infection to reveal novel immune evasion strategies of S. aureus, so as to provide a theoretical basis for developing new anti-S. aureus therapeutic strategies.【Method】Small interfering RNA (siRNA) technology was used to establish a GBP2-knockdown model in the murine macrophage cell line RAW264.7. After S. aureus infection, the expression levels of the target proteins in macrophages were analyzed by western blotting. These proteins included GBP2, key apoptosis-related proteins (Cleaved-Caspase3 and Cleaved-PARP1), regulatory proteins (Bax and Bcl-2), and key molecules of the MAPK signaling pathway (total JNK, ERK, p38, and their phosphorylated forms p-JNK, p-ERK, and p-p38). Changes in GBP2 mRNA expression levels were measured using quantitative real-time polymerase chain reaction. The intracellular localization and expression of GBP2 were observed using immunofluorescence staining. Apoptosis rates were quantified by flow cytometry using the Annexin V-FITC/PI double-staining assay. All experiments included appropriate controls. Differences between groups were analyzed using one-way analysis of variance, with statistical significance set at P<0.05.【Result】S. aureus infection significantly increased both GBP2 protein and mRNA expression levels in RAW264.7 macrophages (P<0.001). Concurrently, infection activated the intrinsic apoptotic pathway, as evidenced by significantly increased expression of Cleaved-Caspase3 and Cleaved-PARP1 (P<0.001), upregulation of the pro-apoptotic protein Bax, downregulation of the anti-apoptotic protein Bcl-2, and a significantly elevated Bax/Bcl-2 ratio (P<0.001). Flow cytometry confirmed a significantly higher apoptosis rate in the S. aureus-infected group than that in the control group (P<0.001). Furthermore, S. aureus infection markedly activated the MAPK signaling pathway, as indicated by significantly increased levels of p-JNK, p-ERK, and p-p38 (P<0.001). When compared with S. aureus infection alone, the siRNA-GBP2+S. aureus group exhibited significant inhibition of the apoptotic phenotype; Cleaved-Caspase3 and Cleaved-PARP1 expression levels were markedly reduced (P<0.001), the Bax/Bcl-2 ratio was significantly decreased (P<0.001), and the apoptosis rates were also significantly reduced (P<0.001). Simultaneously, GBP2 knockdown significantly suppressed the activation of the MAPK pathway, leading to reduced phosphorylation of JNK, ERK, and p38 (P<0.001).【Conclusion】GBP2 acted as a regulator of S. aureus-induced macrophage apoptosis, likely through modulation of MAPK pathway activation. These findings enhanced our understanding of S. aureus pathogenesis and host-pathogen interactions and reveal a novel function of GBP2 in the immune response to bacterial infection. Targeting GBP2 or its MAPK-mediated apoptotic signaling pathway might represent a promising therapeutic approach to combat S. aureus infection, protect host immune cells, and enhance pathogen clearance.

Key words: macrophage, guanylate-binding protein 2 (GBP2), Staphylococcus aureus, MAPK signaling pathway, apoptosis