Laboratório de Microbiologia Molecular e Clínica, Universidade São Francisco, Bragança Paulista, SP, Brazil.
Central Multiusuária de Análises Genômica e Transcriptômica (CmAGT), Universidade São Francisco, Bragança Paulista, SP, Brazil.
BMC Microbiol. 2024 Jul 27;24(1):279. doi: 10.1186/s12866-024-03418-x.
Klebsiella pneumoniae is a Gram-negative pathogen that has become a threat to public health worldwide due to the emergence of hypervirulent and multidrug-resistant strains. Cell-surface components, such as polysaccharide capsules, fimbriae, and lipopolysaccharides (LPS), are among the major virulence factors for K. pneumoniae. One of the genes involved in LPS biosynthesis is the uge gene, which encodes the uridine diphosphate galacturonate 4-epimerase enzyme. Although essential for the LPS formation in K. pneumoniae, little is known about the mechanisms that regulate the expression of uge. Ferric uptake regulator (Fur) is an iron-responsive transcription factor that modulates the expression of capsular and fimbrial genes, but its role in LPS expression has not yet been identified. This work aimed to investigate the role of the Fur regulator in the expression of the K. pneumoniae uge gene and to determine whether the production of LPS by K. pneumoniae is modulated by the iron levels available to the bacterium.
Using bioinformatic analyses, a Fur-binding site was identified on the promoter region of the uge gene; this binding site was validated experimentally through Fur Titration Assay (FURTA) and DNA Electrophoretic Mobility Shift Assay (EMSA) techniques. RT-qPCR analyses were used to evaluate the expression of uge according to the iron levels available to the bacterium. The iron-rich condition led to a down-regulation of uge, while the iron-restricted condition resulted in up-regulation. In addition, LPS was extracted and quantified on K. pneumoniae cells subjected to iron-replete and iron-limited conditions. The iron-limited condition increased the amount of LPS produced by K. pneumoniae. Finally, the expression levels of uge and the amount of the LPS were evaluated on a K. pneumoniae strain mutant for the fur gene. Compared to the wild-type, the strain with the fur gene knocked out presented a lower LPS amount and an unchanged expression of uge, regardless of the iron levels.
Here, we show that iron deprivation led the K. pneumoniae cells to produce higher amount of LPS and that the Fur regulator modulates the expression of uge, a gene essential for LPS biosynthesis. Thus, our results indicate that iron availability modulates the LPS biosynthesis in K. pneumoniae through a Fur-dependent mechanism.
肺炎克雷伯菌是一种革兰氏阴性病原体，由于其产生高毒力和多药耐药菌株，已成为全球公共卫生的威胁。细胞表面成分，如多糖荚膜、菌毛和脂多糖（LPS），是肺炎克雷伯菌主要的毒力因子之一。参与 LPS 生物合成的基因之一是 uge 基因，它编码尿苷二磷酸半乳糖 4-差向异构酶。尽管 ure 基因对于肺炎克雷伯菌 LPS 的形成是必不可少的，但对于调节 ure 表达的机制知之甚少。铁摄取调节因子（Fur）是一种铁反应转录因子，可调节荚膜和菌毛基因的表达，但它在 LPS 表达中的作用尚未确定。本研究旨在探讨 Fur 调节因子在肺炎克雷伯菌 uge 基因表达中的作用，并确定肺炎克雷伯菌 LPS 的产生是否受细菌可用铁水平的调节。
通过生物信息学分析，在 uge 基因启动子区域鉴定到一个 Fur 结合位点；通过 Fur 滴定测定（FURTA）和 DNA 电泳迁移率变动分析（EMSA）技术实验验证了该结合位点的存在。根据细菌可用的铁水平，使用 RT-qPCR 分析评估 uge 的表达。富铁条件导致 uge 下调，而缺铁条件导致 uge 上调。此外，在铁充足和铁限制条件下提取和定量肺炎克雷伯菌细胞的 LPS。铁限制条件增加了肺炎克雷伯菌产生的 LPS 量。最后，在 fur 基因缺失的肺炎克雷伯菌菌株上评估 uge 的表达水平和 LPS 的量。与野生型相比，fur 基因敲除的菌株无论铁水平如何，其 LPS 量较低，而 uge 的表达水平不变。
本研究表明，缺铁导致肺炎克雷伯菌细胞产生更多的 LPS，而 Fur 调节因子调节 ure 基因的表达，ure 基因对于 LPS 生物合成是必不可少的。因此，我们的结果表明，铁的可用性通过 Fur 依赖的机制调节肺炎克雷伯菌的 LPS 生物合成。
