Leuzzi Adriano, Di Martino Maria Letizia, Campilongo Rosaria, Falconi Maurizio, Barbagallo Marialuisa, Marcocci Lucia, Pietrangeli Paola, Casalino Mariassunta, Grossi Milena, Micheli Gioacchino, Colonna Bianca, Prosseda Gianni
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Via dei Sardi 70, 00185, Roma, Italy.
Laboratorio di Genetica Molecolare e dei Microrganismi, Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, Via Gentile III da Varano, Camerino, Italy.
PLoS One. 2015 Aug 27;10(8):e0136744. doi: 10.1371/journal.pone.0136744. eCollection 2015.
The polyamine profile of Shigella, the etiological agent of bacillary dysentery in humans, differs markedly from that of E. coli, its innocuous commensal ancestor. Pathoadaptive mutations such as the loss of cadaverine and the increase of spermidine favour the full expression of the virulent phenotype of Shigella. Spermidine levels affect the expression of the MdtJI complex, a recently identified efflux pump belonging to the small multi-drug resistance family of transporters. In the present study, we have addressed the regulation of the mdtJI operon in Shigella by asking which factors influence its expression as compared to E. coli. In particular, after identifying the mdtJI promoter by primer extension analysis, in vivo transcription assays and gel-retardation experiments were carried out to get insight on the silencing of mdtJI in E. coli. The results indicate that H-NS, a major nucleoid protein, plays a key role in repressing the mdtJI operon by direct binding to the regulatory region. In the Shigella background mdtJI expression is increased by the high levels of spermidine typically found in this microorganism and by VirF, the plasmid-encoded regulator of the Shigella virulence regulatory cascade. We also show that the expression of mdtJI is stimulated by bile components. Functional analyses reveal that MdtJI is able to promote the excretion of putrescine, the spermidine precursor. This leads us to consider the MdtJI complex as a possible safety valve allowing Shigella to maintain spermidine to a level optimally suited to survival within infected macrophages and, at the same time, prevent toxicity due to spermidine over-accumulation.
志贺氏菌是人类细菌性痢疾的病原体，其多胺谱与无害的共生祖先大肠杆菌明显不同。诸如尸胺缺失和亚精胺增加等致病适应性突变有利于志贺氏菌毒力表型的充分表达。亚精胺水平影响MdtJI复合物的表达，MdtJI是最近鉴定出的属于小多药耐药转运蛋白家族的一种外排泵。在本研究中，我们通过询问与大肠杆菌相比哪些因素影响其表达来探讨志贺氏菌中mdtJI操纵子的调控。特别是，通过引物延伸分析鉴定出mdtJI启动子后，进行了体内转录分析和凝胶阻滞实验，以深入了解大肠杆菌中mdtJI的沉默情况。结果表明，主要的类核蛋白H-NS通过直接结合调控区域在抑制mdtJI操纵子中起关键作用。在志贺氏菌背景下，mdtJI的表达因该微生物中通常发现的高水平亚精胺以及志贺氏菌毒力调控级联反应的质粒编码调节因子VirF而增加。我们还表明，胆汁成分可刺激mdtJI的表达。功能分析表明，MdtJI能够促进腐胺（亚精胺前体）的排泄。这使我们认为MdtJI复合物可能是一种安全阀，使志贺氏菌能够将亚精胺维持在最适合在感染巨噬细胞内存活的水平，同时防止由于亚精胺过度积累而产生的毒性。
