School of Chinese Medicine, China Medical University, Taichung, Taiwan.
BMC Microbiol. 2012 Jul 24;12:148. doi: 10.1186/1471-2180-12-148.
The capsular polysaccharide (CPS) and iron acquisition systems are important determinants of Klebsiella pneumoniae infections, and we have previously reported that the ferric uptake repressor (Fur) can play dual role in iron acquisition and CPS biosynthesis. In many bacteria, Fur negatively controls the transcription of the small non-coding RNA RyhB to modulate cellular functions and virulence. However, in K. pneumoniae, the role played by RyhB in the Fur regulon has not been characterised. This study investigated Fur regulation of ryhB transcription and the functional role of RyhB in K. pneumoniae.
Deletion of fur from K. pneumoniae increased the transcription of ryhB; the electric mobility shift assay and the Fur-titration assay revealed that Fur could bind to the promoter region of ryhB, suggesting that Fur directly represses ryhB transcription. Additionally, in a Δfur strain with elevated CPS production, deletion of ryhB obviously reduced CPS production. The following promoter-reporter assay and quantitative real-time PCR of cps genes verified that RyhB activated orf1 and orf16 transcription to elevate CPS production. However, deletion of ryhB did not affect the mRNA levels of rcsA, rmpA, or rmpA2. These results imply that Fur represses the transcription of ryhB to mediate the biosynthesis of CPS, which is independent of RcsA, RmpA, and RmpA2. In addition, the Δfur strain's high level of serum resistance was attenuated by the deletion of ryhB, indicating that RyhB plays a positive role in protecting the bacterium from serum killing. Finally, deletion of ryhB in Δfur reduced the expression of several genes corresponding to 3 iron acquisition systems in K. pneumoniae, and resulted in reduced siderophore production.
The regulation and functional role of RyhB in K. pneumoniae is characterized in this study. RyhB participates in Fur regulon to modulate the bacterial CPS biosynthesis and iron acquisition systems in K. pneumoniae.
荚膜多糖（CPS）和铁摄取系统是肺炎克雷伯菌感染的重要决定因素，我们之前曾报道过，三价铁摄取阻遏物（Fur）在铁摄取和 CPS 生物合成中可以发挥双重作用。在许多细菌中，Fur 负调控小非编码 RNA RyhB 的转录，以调节细胞功能和毒力。然而，在肺炎克雷伯菌中，RyhB 在 Fur 调控中的作用尚未得到表征。本研究探讨了 Fur 对 ryhB 转录的调控作用以及 RyhB 在肺炎克雷伯菌中的功能作用。
肺炎克雷伯菌 fur 缺失增加了 ryhB 的转录；电泳迁移率变动分析和 Fur 滴定分析表明，Fur 可以结合到 ryhB 的启动子区域，表明 Fur 直接抑制 ryhB 转录。此外，在 CPS 产量升高的Δfur 菌株中，ryhB 的缺失明显降低了 CPS 产量。随后的启动子报告基因和 cps 基因的定量实时 PCR 验证了 RyhB 激活 orf1 和 orf16 的转录以提高 CPS 产量。然而，ryhB 的缺失并不影响 rcsA、rmpA 或 rmpA2 的 mRNA 水平。这些结果表明，Fur 抑制 ryhB 的转录以介导 CPS 的生物合成，这与 RcsA、RmpA 和 RmpA2 无关。此外，ryhB 的缺失削弱了Δfur 菌株对血清的高抗性，表明 RyhB 在保护细菌免受血清杀伤方面发挥积极作用。最后，ryhB 在Δfur 中的缺失降低了肺炎克雷伯菌中 3 个铁摄取系统的几个基因的表达，导致铁载体产量减少。
本研究对 RyhB 在肺炎克雷伯菌中的调控和功能作用进行了描述。RyhB 参与 Fur 调控，调节肺炎克雷伯菌的细菌 CPS 生物合成和铁摄取系统。
