Deciphering FUR-regulated gene networks in Klebsiella pneumoniae using FUR knockout mutant

The Microbe Pub Date : 2024-11-08 DOI:10.1016/j.microb.2024.100201

Purvi Joshi , Reetu Maindolia , Dhanshree Dhakre, Twinkle Patel, Devarshi Gajjar

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Abstract

Klebsiella pneumoniae is a nosocomial pathogen causing life-threatening infections worldwide. It possesses many virulence properties and the ferric uptake regulator (FUR) is the transcription factor that regulates the expression of various genes in K. pneumoniae. FUR binds to a conserved 19 bp sequence, called the FUR box, for either positive or negative regulation of the gene. FUR alters gene expression according to iron availability and also participates in virulence, colonization & toxin secretion in K. pneumoniae. FUR deletion mutant is an interesting model to understand the virulence factors. In the present study, we identified the genes containing putative FUR boxes (>50 % sequence homology) from the genome of K. pneumoniae M33. FUR deletion mutant for K. pneumoniae M33 was created using lambda red recombinase system using the plasmids- pACBSR-hyg, pMDIAI, and pFLP-hyg. The mutant (M33∆FUR) was characterized using various assays like CAS assay, string test, crystal violet assay to access siderophore production, capsule synthesis and biofilm formation respectively. FUR box was present upstream of 18 genes with distinct functions. In the FUR knockout mutant, M33∆FUR an increase in expression of iron transport and siderophore related genes feoC, fhuA, fepB and fes was observed whereas genes feoA, cirA, fecA, fepA and entC were down-regulated. Genes related to biofilm (fimA and mrkD) were downregulated whereas genes related to capsular polysaccharide (rcsA and rcsB) were upregulated upon FUR deletion. Among other genes murG and sucA were downregulated and priB and srlB were upregulated upon FUR deletion. This study shows that FUR regulates many genes involved in virulence, either positively or negatively.

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利用 FUR 基因敲除突变体破译肺炎克雷伯菌中的 FUR 调控基因网络

肺炎克雷伯氏菌（Klebsiella pneumoniae）是一种引起全球范围内危及生命感染的医院病原体。它具有许多毒力特性，铁吸收调节因子（FUR）是调节肺炎克雷伯氏菌各种基因表达的转录因子。FUR 与一个名为 FUR 框的 19 bp 保守序列结合，对基因进行正向或负向调节。FUR 根据铁的可用性改变基因表达，还参与肺炎双球菌的毒力、定植和毒素分泌。FUR 缺失突变体是了解毒力因子的一个有趣模型。在本研究中，我们从肺炎克雷伯菌 M33 的基因组中鉴定出了含有推定 FUR 框（50% 序列同源性）的基因。利用λ红重组酶系统，使用质粒 pACBSR-hyg、pMDIAI 和 pFLP-hyg，创建了肺炎双球菌 M33 的 FUR 缺失突变体。突变体（M33∆FUR）通过 CAS 试验、串联试验、水晶紫试验等多种检测方法分别鉴定了嗜苷酸盐的产生、胶囊的合成和生物膜的形成。FUR 框存在于具有不同功能的 18 个基因的上游。在 FUR 基因敲除突变体 M33∆FUR 中，观察到铁转运和嗜苷酸盐相关基因 feoC、fhuA、fepB 和 fes 的表达增加，而基因 feoA、cirA、fecA、fepA 和 entC 的表达下调。删除 FUR 后，与生物膜相关的基因（fimA 和 mrkD）下调，而与胶囊多糖相关的基因（rcsA 和 rcsB）上调。在其他基因中，FUR缺失后，murG和sucA下调，priB和srlB上调。这项研究表明，FUR 对许多涉及毒力的基因具有正向或负向调控作用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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The Microbe

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