MLKL对金黄色葡萄球菌诱导的炎症介质分泌及炎症信号通路激活的影响

doi:10.16431/j.cnki.1671-7236.2024.12.042

Abstract

Abstract: 【Objective】 Staphylococcus aureus (S.aureus),a Gram-positive facultative anaerobic bacterium,was an intracellular pathogen capable of causing various diseases in both humans and animal hosts through the utilization of necrotic apoptosis.This study was aimed to investigate whether the mixed lineage kinase domain-like protein (MLKL) was involved in regulating the inflammatory response induced by S.aureus infection,and provide new ideas and theoretical basis for the prevention and control of diseases caused by S.aureus infection.【Method】 In this study,genetic change in mouse macrophages before and after stimulation with Pam2CSK4 and S.aureus SA113 were analyzed by transcriptome sequencing.The docking modes and sites of Pam2CSK4 and MLKL were analyzed by molecular docking technique.The differentially expressed genes were verified by Real-time quantitative PCR.The phosphorylation levels of MLKL,p65,p38 and ERK were detected by Western blotting.The secretion levels of pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)),chemokine (RANTES) and anti-inflammatory factor (IL-10) were detected by ELISA.【Result】 The results of transcriptional analysis showed that stimulation with Pam2CSK4 and S.aureus SA113 induced the expression of necroptosis-related genes in macrophages,with a high correlation in MLKL gene(P＜0.05).Molecular docking prediction results showed that Pam2CSK4 was well bound to MLKL protein and formed hydrogen bonds with ARG-304,ARG-301,GLN-331 and LYS-333 amino acids.Real-time quantitative PCR and Western blotting results showed that compared to unstimulated group, the stimulation with Pam2CSK4 and S.aureus SA113 led to an extremely significant upregulation in the phosphorylation and gene expression levels of MLKL in mouse macrophages (P＜0.01).These findings suggested the involvement of MLKL in the S.aureus infection process.The impact of MLKL on the activation of the NF-κB and MAPK signaling pathways was examined using Western blotting.The results indicated that compared with C57BL/6J mice,MLKL^-/- mouse macrophages exhibited a significant or extremely significant increase in p65 phosphorylation after S.aureus stimulation for 30 and 60 min (P＜0.05 or P＜0.01),while an extremely significant decrease was observed after Pam2CSK4 stimulation for 30 and 60 min (P＜0.01).After 15,30 and 60 min of Pam2CSK4 stimulation,the phosphorylation level of ERK in MLKL^-/- mouse macrophages was extremely significantly downregulated (P＜0.01).After 60 min of stimulation with Pam2CSK4 and S.aureus SA113,the phosphorylation level of p38 in MLKL^-/- mouse macrophages was significantly or extremely significantly downregulated (P＜0.05 or P＜0.01). Compared with C57BL/6J mice,after Pam2CSK4 stimulation,the secretion levels of TNF-α and RANTES in the supernatant of MLKL^-/- mice macrophages were extremely significantly increased (P＜0.01).After SA113 stimulation,the secretion levels of TNF-α,IL-1β and RANTES in the supernatant of MLKL^-/- mouse macrophages were extremely significantly increased (P＜0.01),while the secretion level of IL-10 was extremely significantly decreased (P＜0.01).【Conclusion】 These results suggested that MLKL played a regulatory role in the secretion of inflammatory mediators and the activation of inflammatory signaling pathways induced by S.aureus.

Key words: Staphylococcus aureus; mixed lineage kinase domain like protein (MLKL); necroptosis; transcriptomics; inflammatory response

CLC Number:

S852.61

LIU Bo, GONG Zhiguo, HASI Surong. Effects of MLKL on Secretion of Inflammatory Mediators and Activation of Inflammatory Signaling Pathway Induced by Staphylococcus aureus[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5560-5569.

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Effects of MLKL on Secretion of Inflammatory Mediators and Activation of Inflammatory Signaling Pathway Induced by Staphylococcus aureus

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