血小板致金黄色葡萄球菌发生自溶的机制探讨

Discussion on the mechanism of platelet-induced autolysis of Staphylococcus aureus

目的探索血小板抗金黄色葡萄球菌(SA)的效能并尝试阐明其抗菌作用机制。方法实验分为SA组和血小板处理组,采用紫外分光光度计测定3次SA组和血小板处理组共6份菌液OD600值及6份菌液涂板菌落计数评价血小板的抗SA效能;分别采用His Sq2000测序仪和Q-Exactive质谱仪做基因转录组学和蛋白质谱分析检测血小板处理金黄色葡萄球菌后在基因转录水平和蛋白水平细菌自溶能力的变化,并通过做qRT-PCR和Trition X-100诱导下细菌自溶能力检测进一步验证。结果菌液OD600值测定试验显示:终浓度(×109/L)为100、150、200的血小板处理SA 10 h后菌液OD600值分别为0.314、0.152、0.234,而对照组为1.223(P<0.01),其中血小板浓度为150×109/L时处理SA后OD600值下降幅度明显大于其他血小板浓度处理组(P<0.01);与对照组SA相比,血小板(150×109/L)处理SA后,在转录水平上调控细菌自溶活性的双组分系统基因lyt S、lytR及其下游基因lrg A、lrg B变化倍数分别为0.165、0.086、0.045、0.107,下调明显(P<0.01),自溶酶基因atl A为3.596,上调明显(P<0.01),在蛋白水平上,蛋白质谱分析SA水解酶和自溶酶相对定量:血小板处理组和对照组分别为1.307 vs 0.633、1.326 vs 0.834(P<0.01);血小板(150×109/L)处理SA后与对照组在Triton X-100的诱导下细菌的浊度(OD600)比较:0.5、1.5、2.5h分别为0.052 vs 0.112、0.046 vs 0.089、0.048 vs 0.061(P<0.01)。结论血小板通过作用于金黄色葡萄球菌双组份系统自溶调节基因致细菌发生自溶来发挥抗菌作用;本研究发现所发现的血小板对于金黄色葡萄球菌新的抑菌靶点和途径,或为临床综合治疗细菌性感染提供新的策略和思路。

Objective To explore the efficacy of platelets against Staphylococcus aureus(SA)and to elucidate the mechanism of its antibacterial action.Methods The experiment was divided into SA group and platelet treatment group.The OD600 values and colony counts of bacterial solutions,sampled for three times in both SA group and platelet treatment group,were measured by UV spectrophotometer to evaluate the anti-SA efficacy of platelets.His Sq2000 sequencing instrument and Q-Exactive mass spectrometer were used for gene transcriptome and protein spectrum analysis to detect the changes of bacterial autolysis capacity at gene transcription level and protein level after platelet treatment of staphylococcus aureus,and further verified by qRT-PCR and Tritionx-100 induction.Results The determination of OD600 values of bacterial solutions showed that:the OD600 values of bacterial solutions after 10 h-treatment with a final platelet concentration(×109/L)of 100,150 and200 was 0.314,0.152 and 0.234,respectively,while that of control group was 1.223(P<0.01).When the platelet concentration was 150×109/L,the OD600 value decreased significantly greater than that of other platelet concentration groups(P<0.01).Compared with the control group,the bicomponent system genes(lyt S and lytR)as well as their downstream genes(lrg A and lrg B)that regulated the bacterial autolytic activity at the transcriptional level were significantly down-regulated after the treatment of platelet(150×109/L),whose fold changes were 0.165,0.086,0.045 and 0.107,respectively(P﹤0.01);while the autolytic gene atl A was significantly up-regulated,with a fold change of 3.596(P﹤0.01).Comparison of the relative quantification of SA hydrolytic enzyme and autolytic enzyme by protein spectrum analysis between platelet treatment group(150×109/L)and control group was 1.307 vs.0.633 and 1.326 vs.0.834,respectively(P<0.01).Comparison of bacterial turbidity(OD600)between control group and platelet treatment group(150×109/L)treated with Triton x-100 for 0.5,1.5 and 2.5 hours were as follows:0.052 vs.0.112,0.046 vs.0.089,and 0.048 vs.0.061,respectively(P<0.01).Conclusion Platelets play an antibacterial role by acting on the bicomponent autolytic regulatory gene of Staphylococcus aureus to induce bacterial autolysis.The findings of this study may provide new strategies and ideas for the comprehensive treatment of bacterial infections.