Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.
Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.
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DOI：
10.1021/acs.biochem.6b00083
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发表时间：
2016-05-03
期刊：
影响因子：
2.9
通讯作者：
Schlievert PM
中科院分区：
文献类型：
--
作者：
Herrera A;Vu BG;Stach CS;Merriman JA;Horswill AR;Salgado-Pabón W;Schlievert PM
β-毒素是金黄色葡萄球菌的重要毒力因子，有助于其定植和疾病的发展。这种细胞毒素具有两种不同的作用机制：鞘磷脂酶活性和DNA生物膜连接酶活性。然而，对其在感染性心内膜炎中的作用重要的独特机制尚不清楚。我们通过体外DNA沉淀和生物膜形成试验检测定点突变体的缺陷，表征了β毒素DNA生物膜连接酶活性的活性位点。与野生型毒素相比，突变体结构中可能的构象变化通过胰蛋白酶消化分析、鞘磷脂酶活性保留和基于天然毒素结构的预测结构进行评估。我们在感染性心内膜炎和脓毒症的家兔模型中，探讨了在体内产生感染性心内膜炎的每种作用机制的作用。H289 N β-毒素S.与野生型蛋白相比，缺乏鞘磷脂酶活性的金黄色葡萄球菌突变体的致死率和感染性心内膜炎赘生物形成降低。生物膜连接酶活性被破坏的β毒素突变体不降低S。金黄色葡萄球菌致死率，但缺乏感染性心内膜炎赘生物形成相比，野生型蛋白。我们的研究开始描述β毒素的DNA生物膜连接酶活性位点，并表明β毒素通过其两种作用机制在感染性心内膜炎中发挥重要作用。
β-toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism which is important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to wild type toxin were assessed via trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action in producing infective endocarditis in vivo in a rabbit model of infective endocarditis and sepsis. The H289N β-toxin S. aureus mutant, lacking sphingomyelinase activity, was decreased in lethality and infective endocarditis vegetation formation compared to wild type protein. β-toxin mutants disrupted in biofilm ligase activity did not decrease S. aureus lethality, but were deficient in infective endocarditis vegetation formation compared to wild type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.