Haines Sara, Arnaud-Barbe Nadège, Poncet David, Reverchon Sylvie, Wawrzyniak Julien, Nasser William, Renauld-Mongénie Geneviève
Research Department, Sanofi Pasteur, Marcy-l'Étoile, France UMR5240 CNRS/INSA/UCB, Université de Lyon, Villeurbanne, France, and INSA de Lyon, Villeurbanne, France.
Research Department, Sanofi Pasteur, Marcy-l'Étoile, France.
J Bacteriol. 2015 Sep;197(18):2896-907. doi: 10.1128/JB.00214-15. Epub 2015 Jun 29.
Iron availability functions as an environmental cue for enteropathogenic bacteria, signaling arrival within the human host. As enterotoxigenic Escherichia coli (ETEC) is a major cause of human diarrhea, the effect of iron on ETEC virulence factors was evaluated here. ETEC pathogenicity is directly linked to production of fimbrial colonization factors and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). Efficient colonization of the small intestine further requires at least the flagellin binding adhesin EtpA. Under iron starvation, production of the CFA/I fimbriae was increased in the ETEC H10407 prototype strain. In contrast, LT secretion was inhibited. Furthermore, under iron starvation, gene expression of the cfa (CFA/I) and etp (EtpBAC) operons was induced, whereas transcription of toxin genes was either unchanged or repressed. Transcriptional reporter fusion experiments focusing on the cfa operon further showed that iron starvation stimulated cfaA promoter activity in ETEC, indicating that the impact of iron on CFA/I production was mediated by transcriptional regulation. Evaluation of cfaA promoter activity in heterologous E. coli single mutant knockout strains identified IscR as the regulator responsible for inducing cfa fimbrial gene expression in response to iron starvation, and this was confirmed in an ETEC ΔiscR strain. The global iron response regulator, Fur, was not implicated. IscR binding sites were identified in silico within the cfaA promoter and fixation confirmed by DNase I footprinting, indicating that IscR directly binds the promoter region to induce CFA/I.
Pathogenic enterobacteria modulate expression of virulence genes in response to iron availability. Although the Fur transcription factor represents the global regulator of iron homeostasis in Escherichia coli, we show that several ETEC virulence factors are modulated by iron, with expression of the major fimbriae under the control of the iron-sulfur cluster regulator, IscR. Furthermore, we demonstrate that the apo form of IscR, lacking an Fe-S cluster, is able to directly fix the corresponding promoter region. These results provide further evidence implicating IscR in bacterial virulence and suggest that IscR may represent a more general regulator mediating the iron response in enteropathogens.
铁的可利用性作为肠道致病菌的一种环境信号，标志着其进入人类宿主。由于产肠毒素大肠杆菌（ETEC）是人类腹泻的主要病因，本文评估了铁对ETEC毒力因子的影响。ETEC的致病性与菌毛定植因子的产生以及热不稳定肠毒素（LT）和/或热稳定肠毒素（ST）的分泌直接相关。小肠的有效定植还至少需要鞭毛蛋白结合黏附素EtpA。在铁饥饿条件下，ETEC H10407原型菌株中CFA/I菌毛的产生增加。相反，LT的分泌受到抑制。此外，在铁饥饿条件下，cfa（CFA/I）和etp（EtpBAC）操纵子的基因表达被诱导，而毒素基因的转录要么未改变，要么受到抑制。针对cfa操纵子的转录报告融合实验进一步表明，铁饥饿刺激了ETEC中cfaA启动子的活性，这表明铁对CFA/I产生的影响是由转录调控介导的。在异源大肠杆菌单突变敲除菌株中评估cfaA启动子活性，确定IscR是负责响应铁饥饿诱导cfa菌毛基因表达的调节因子，这在ETEC ΔiscR菌株中得到了证实。全局铁反应调节因子Fur未涉及其中。通过计算机模拟在cfaA启动子内鉴定出IscR结合位点，并通过DNase I足迹法证实了其结合，表明IscR直接结合启动子区域以诱导CFA/I。
致病性肠道细菌会根据铁的可利用性调节毒力基因的表达。虽然Fur转录因子是大肠杆菌中铁稳态的全局调节因子，但我们表明几种ETEC毒力因子受铁调节，主要菌毛的表达受铁硫簇调节因子IscR的控制。此外，我们证明缺乏Fe-S簇的IscR脱辅基形式能够直接结合相应的启动子区域。这些结果为IscR参与细菌毒力提供了进一步证据，并表明IscR可能代表一种更普遍的调节因子，介导肠道病原体中的铁反应。
