School of Public Health, Chongqing Medical University, Chongqing, China.
Chengdu Center for Disease Control and Prevention, Chengdu, Sichuan, China.
J Basic Microbiol. 2024 Aug;64(8):e2400001. doi: 10.1002/jobm.202400001. Epub 2024 Apr 28.
The ferric uptake regulator (Fur) is a global regulator that influences the expression of virulence genes in Klebsiella pneumoniae. Bioinformatics analysis suggests Fur may involve in iron acquisition via the identified regulatory box upstream of the yersiniabactin receptor gene fyuA. To observe the impact of the gene fyuA on the virulence of K. pneumoniae, the gene fyuA knockout strain and complementation strain were constructed and then conducted a series of phenotypic experiments including chrome azurol S (CAS) detection, crystal violet staining, and wax moth virulence experiment. To examine the regulatory relationship between Fur and the gene fyuA, green fluorescent protein (GFP) reporter gene fusion assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), gel migration assay (EMSA), and DNase I footprinting assay were used to clarify the regulatory mechanism of Fur on fyuA. CAS detection revealed that the gene fyuA could affect the generation of iron carriers in K. pneumoniae. Crystal violet staining experiment showed that fyuA could positively influence biofilm formation. Wax moth virulence experiment indicated that the deletion of the fyuA could weaken bacterial virulence. GFP reporter gene fusion experiment and RT-qPCR analysis revealed that Fur negatively regulated the expression of fyuA in iron-sufficient environment. EMSA experiment demonstrated that Fur could directly bind to the promoter region of fyuA, and DNase I footprinting assay further identified the specific binding site sequences. The study showed that Fur negatively regulated the transcriptional expression of fyuA by binding to upstream of the gene promoter region, and then affected the virulence of K. pneumoniae.
铁摄取调节因子（Fur）是一种全局调节因子，影响肺炎克雷伯菌毒力基因的表达。生物信息学分析表明，Fur 可能通过鉴定的yersiniabactin 受体基因 fyuA 上游的调节框参与铁的摄取。为了观察基因 fyuA 对肺炎克雷伯菌毒力的影响，构建了基因 fyuA 敲除株和互补株，并进行了一系列表型实验，包括铬天青 S（CAS）检测、结晶紫染色和黄粉虫毒力实验。为了研究 Fur 和基因 fyuA 之间的调控关系，使用绿色荧光蛋白（GFP）报告基因融合测定、实时定量逆转录聚合酶链反应（RT-qPCR）、凝胶迁移测定（EMSA）和 DNase I 足迹测定来阐明 Fur 对 fyuA 的调控机制。CAS 检测表明，基因 fyuA 可以影响肺炎克雷伯菌中铁载体的产生。结晶紫染色实验表明，fyuA 可以正向影响生物膜的形成。黄粉虫毒力实验表明，fyuA 的缺失会削弱细菌的毒力。GFP 报告基因融合实验和 RT-qPCR 分析表明，在铁充足的环境中，Fur 负调控 fyuA 的表达。EMSA 实验表明 Fur 可以直接结合到 fyuA 的启动子区域，DNase I 足迹测定进一步鉴定了特定的结合位点序列。该研究表明，Fur 通过结合基因启动子区域的上游，负调控 fyuA 的转录表达，从而影响肺炎克雷伯菌的毒力。
