CcpA Regulates Staphylococcus aureus Biofilm Formation through Direct Repression of Staphylokinase Expression.
CcpA Regulates Staphylococcus aureus Biofilm Formation through Direct Repression of Staphylokinase Expression.
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CcpA 通过直接抑制葡萄球菌激酶表达来调节金黄色葡萄球菌生物膜形成
DOI：
10.3390/antibiotics11101426
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发表时间：
2022-10-17
期刊：
影响因子：
--
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中科院分区：
文献类型：
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金黄色葡萄球菌是一种臭名昭著的机会性病原体，可引起生物被膜性质的各种感染，在全球范围内构成了显著的治疗挑战。分解代谢控制蛋白A(CCPA)是碳分解代谢抑制(CCR)的主要调节因子，对金黄色葡萄球菌生物膜的形成具有重要的调控作用，但其作用机制尚不清楚。本研究首次用结晶紫染色和激光共聚焦扫描显微镜检测了金黄色葡萄球菌临床分离株XN108 CCPA缺失突变株的生物膜还原情况。Rna-seq分析表明，突变株∆ccpA的SAK编码蛋白(SAK)显著上调，RT-qPCR进一步证实了这一点。PSAK-LacZ融合表达和显色检测表明，SAK的诱导产生与SAK启动子活性的提高和培养上清液分泌的增加密切相关。值得注意的是，电泳迁移率改变分析表明，纯化的重组蛋白CCPA直接与SAK的启动子区域结合，表明CCPA直接负调控SAK的表达。CcpA和sak的双重等基因缺失恢复了突变体∆ccpA的生物膜形成，反式互补的sak可以减少这种形成。此外，外源重组SAK的加入抑制了XN108生物膜的形成，且呈剂量依赖关系。总之，本研究通过直接抑制sak的表达，描述了CCPA控制的金黄色葡萄球菌生物被膜的新模型，突出了CCPA调控的多方面的作用和多个网络。
Staphylococcus aureus represents a notorious opportunistic pathogen causing various infections in biofilm nature, imposing remarkable therapeutic challenges worldwide. The catabolite control protein A (CcpA), a major regulator of carbon catabolite repression (CCR), has been recognized to modulate S. aureus biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the ccpA deletion mutant of S. aureus clinical isolate XN108 using both crystal violet staining and confocal laser scanning microscopy. RNA-seq analysis suggested that sak-encoding staphylokinase (Sak) was significantly upregulated in the mutant ∆ccpA, which was further confirmed by RT-qPCR. Consistently, the induced Sak production correlated the elevated promoter activity of sak and increased secretion in the supernatants, as demonstrated by Psak-lacZ reporter fusion expression and chromogenic detection, respectively. Notably, electrophoretic mobility shift assays showed that purified recombinant protein CcpA binds directly to the promoter region of sak, suggesting the direct negative control of sak expression by CcpA. Double isogenic deletion of ccpA and sak restored biofilm formation for mutant ∆ccpA, which could be diminished by trans-complemented sak. Furthermore, the exogenous addition of recombinant Sak inhibited biofilm formation for XN108 in a dose-dependent manner. Together, this study delineates a novel model of CcpA-controlled S. aureus biofilm through direct inhibition of sak expression, highlighting the multifaceted roles and multiple networks regulated by CcpA.
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