金黄色葡萄球菌分泌蛋白LukG抑制巨噬细胞炎症反应

Staphylococcus aureus LukG suppress the inflammatory responses of macrophages

目的利用同源重组法构建金黄色葡萄球菌LukG(leukotoxin G)基因敲除株,探究LukG缺失对金黄色葡萄球菌刺激巨噬细胞炎症反应的影响。方法用双荧光素酶报告基因检测LukG蛋白对NF-κB通路的作用;利用USA300野生株和LukG敲除株(ΔLukG/USA300)感染小鼠原代腹腔巨噬细胞,实时荧光定量PCR法检测TNF-α、IL-6和IL-1β的mRNA转录水平,Western blot检测IκB-α及p-p65表达水平;利用USA300和ΔLukG/USA300气管插管感染小鼠肺部,观察生存率差异,稀释涂板法测定肺部荷菌量,HE染色法观察肺组织病理学变化,ELISA法检测肺组织匀浆炎性细胞因子TNF-α、IL-6和IL-1β的表达量。结果分泌蛋白LukG能够明显抑制NF-κB通路的激活;感染原代腹腔巨噬细胞后;相对于USA300感染组,ΔLukG/USA300感染组TNF-α、IL-6和IL-1β的转录水平升高(P<0.05),IκB-α表达下调,p-p65表达上调;气管插管感染小鼠后,相对于USA300感染组,ΔLukG/USA300感染组生存率提高(P<0.05),肺组织荷菌量降低(P<0.05),病变程度减轻,炎性细胞因子TNF-α、IL-6和IL-1β表达量升高(P<0.05)。结论金黄色葡萄球菌可能通过分泌LukG蛋白抑制巨噬细胞NF-κB通路进而下调炎性细胞因子的表达,达成免疫逃逸进而成功感染宿主。

This study aimed to explore the effects of Staphylococcus aureus(S.aureus)LukG on the inflammatory responses of macrophages.The effect of LukG protein on NF-κB pathway was detected by dual luciferase reporter gene assay.After primary peritoneal macrophages were infected by USA300 strain orΔLukG/USA300 strain,qRT-PCR was used to detect the transcription levels of TNF-α,IL-6 and IL-1β,while Western blot was used to detet the expression of IκB-αand p-p65.After mice were infected by USA300 strain orΔLukG/USA300 strain through endotracheal intubation,the survival rates were recorded daily.The amount of bacteria in the lung of mice was measured by the spread plate method;the pathological changes of lung tissue were observed by HE staining;ELISA was used to detect the expression of inflammatory cytokines TNF-α,IL-6 and IL-1βin the lung tissue homogenate.The results of double luciferase reporter gene showed that LukG could suppress the activation of NF-κB pathway.Compared with those in the USA300 strain infection group,the transcription levels of TNF-α,IL-6 and IL-1βwere increased(P<0.05),the IκB-αand p-p65 expression levels were respectively down-regulated and up-regulated inΔLukG/USA300 strain infection group.Compared with the USA300 strain infection group,theΔLukG/USA300 strain infection group demonstrated higher survival rates,lower bacterial loads,less pathological changes and higher expression of TNF-α,IL-6 and IL-1β(P<0.05).Taken together,Staphylococcus aureus could secrete LukG to inhibit the NF-κB pathway of macrophages and down-regulate the expression of inflammatory cytokines.This may be the strategy used by Staphylococcus aureus to achieve immune escape and infect the host successfully.