Zheng Mingxia, Zhu Keting, Peng Huagang, Shang Weilong, Zhao Yan, Lu Shuguang, Rao Xiancai, Li Ming, Zhou Renjie, Li Gang
Department of Emergency Medicine, Xinqiao Hospital, Army Medical University, Chongqing 400037, China.
Department of Microbiology, College of Basic Medical Sciences, Army Medical University, Chongqing 400038, China.
Antibiotics (Basel). 2022 Oct 17;11(10):1426. doi: 10.3390/antibiotics11101426.
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 biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the deletion mutant of clinical isolate XN108 using both crystal violet staining and confocal laser scanning microscopy. RNA-seq analysis suggested that -encoding staphylokinase (Sak) was significantly upregulated in the mutant ∆, which was further confirmed by RT-qPCR. Consistently, the induced Sak production correlated the elevated promoter activity of and increased secretion in the supernatants, as demonstrated by P- 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 , suggesting the direct negative control of expression by CcpA. Double isogenic deletion of and restored biofilm formation for mutant ∆, which could be diminished by -complemented . 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 biofilm through direct inhibition of expression, highlighting the multifaceted roles and multiple networks regulated by CcpA.
代表一种臭名昭著的机会致病菌，在生物膜状态下可引起各种感染，给全球带来了严峻的治疗挑战。分解代谢物控制蛋白A（CcpA）是碳分解代谢物阻遏（CCR）的主要调节因子，已被认为可调节生物膜形成，但其潜在机制仍有待充分阐明。在本研究中，首先使用结晶紫染色和共聚焦激光扫描显微镜在临床分离株XN108的缺失突变体中确定了生物膜的减少。RNA测序分析表明，编码葡萄球菌激酶（Sak）的基因在突变体∆中显著上调，这通过RT-qPCR得到进一步证实。一致地，诱导的Sak产生与基因启动子活性的升高以及上清液中分泌的增加相关，分别通过P-报告基因融合表达和显色检测得以证明。值得注意的是，电泳迁移率变动分析表明，纯化的重组蛋白CcpA直接结合到基因的启动子区域，表明CcpA对基因表达具有直接的负调控作用。对突变体∆进行基因和基因的双同源缺失可恢复生物膜形成，而这可被基因互补所减弱。此外，外源添加重组Sak以剂量依赖的方式抑制XN108的生物膜形成。总之，本研究通过直接抑制基因表达描绘了一种CcpA控制生物膜的新模型，突出了CcpA的多方面作用和多个调控网络。
