GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms.
GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms.
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DOI：
10.15698/mic2023.07.800
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发表时间：
2023-07-03
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金黄色葡萄球菌是一种主要的人类病原体，它利用许多表面相关和分泌的蛋白质形成生物膜并引起疾病。然而，我们对这些过程的理解受到在其天然环境中使用荧光蛋白报告分子的挑战的限制，因为它们必须被正确地导出和折叠才能成为荧光蛋白。在这里，我们证明了使用从S.金黄色。通过将msfGFP与分泌（Sec）和双精氨酸转位（达特）途径的信号肽融合，S.金黄色葡萄球菌，我们定量了细菌培养物和来自培养物的无细胞上清液中的msfGFP荧光。当与达特信号肽融合时，我们在细菌细胞内检测到msfGFP荧光，但在细菌细胞外检测不到，表明msfGFP输出失败。然而，当与Sec信号肽融合时，msfGFP荧光存在于细胞外，表明未折叠状态的msfGFP成功输出，随后细胞外折叠并成熟为光活性状态。我们应用这种策略来研究凝固酶（CoA），一种分泌蛋白，是S.金黄色葡萄球菌生物膜，保护细菌免受宿主免疫系统的侵害，并增加对宿主表面的附着。我们证实，基因组整合的C-末端融合的CoA msfGFP不损害CoA的活性或其在生物膜基质内的定位。我们的研究结果表明，msfGFP是一个很好的候选荧光报告时，考虑在S.金黄色。
Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluorescent protein reporters in their native environment, because they must be exported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.