PDF(Pubmed)
Abstract：
Successful colonization by the opportunistic pathogen Staphylococcus aureus depends on its ability to interact with other microorganisms. Staphylococcus aureus strains harbour a T7b subtype of type VII secretion system (T7SSb), a protein secretion system found in a wide variety of Bacillota, which functions in bacterial antagonism and virulence. Assessment of T7SSb activity in S. aureus has been hampered by low secretion activity under laboratory conditions and the lack of a sensitive assay to measure secretion. Here, we have utilized NanoLuc binary technology to develop a simple assay to monitor protein secretion via detection of bioluminescence. Fusion of the 11 amino acid NanoLuc fragment to the conserved substrate EsxA permits its extracellular detection upon supplementation with the large NanoLuc fragment and luciferase substrate. Following miniaturization of the assay to 384-well format, we use high-throughput analysis to demonstrate that T7SSb-dependent protein secretion differs across strains and growth temperature. We further show that the same assay can be used to monitor secretion of the surface-associated toxin substrate TspA. Using this approach, we identify three conserved accessory proteins required to mediate TspA secretion. Co-purification experiments confirm that all three proteins form a complex with TspA.
摘要：
机会病原体金黄色葡萄球菌的成功定殖取决于其与其他微生物相互作用的能力。金黄色葡萄球菌菌株具有VII型分泌系统（T7SSb）的T7b亚型，一种在各种芽孢杆菌中发现的蛋白质分泌系统，在细菌拮抗和毒力中起作用。金黄色葡萄球菌中T7SSb活性的评估受到实验室条件下的低分泌活性和缺乏测量分泌的灵敏测定法的阻碍。这里,我们利用NanoLuc二元技术开发了一种简单的检测方法，通过检测生物发光来监测蛋白质分泌。11个氨基酸的NanoLuc片段与保守底物EsxA的融合允许其在补充大NanoLuc片段和荧光素酶底物后的细胞外检测。在将测定小型化为384孔格式之后，我们使用高通量分析来证明T7SSb依赖性蛋白分泌在菌株和生长温度之间存在差异.我们进一步显示相同的测定可用于监测表面相关毒素底物TspA的分泌。使用这种方法，我们鉴定了介导TspA分泌所需的三种保守的辅助蛋白。共纯化实验证实所有三种蛋白质与TspA形成复合物。
