Integration host factor is involved in transcriptional regulation of the Brucella abortus virB operon
Integration host factor is involved in transcriptional regulation of the Brucella abortus virB operon
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DOI：
10.1111/j.1365-2958.2004.04316.x
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发表时间：
2004-11-01
影响因子：
3.6
通讯作者：
Ugalde, RA
中科院分区：
文献类型：
--
作者：
Sieira, R;Comerci, DJ;Ugalde, RA
IV型分泌系统（T4 SSs）是在许多兼性胞内细菌的致病性中起重要作用的多组分机制。牛种布氏杆菌的virB操纵子编码毒力和细胞内增殖所必需的T4 SS。在此，使用lacZ转录融合进行的virB表达分析显示，virB启动子（P-virB）在J774细胞内被暂时激活。引物延伸实验表明，virB转录开始于virB操纵子的第一个基因上游27 bp处。结构分析表明，P-virB和调控序列参与细胞内调控跨度430 bp的转录起始位点上游。分离并鉴定了能够结合P-virB的蛋白。该蛋白与整合宿主因子（IHF）同源，能特异性地与P-virB相互作用，诱导DNA发生50.36 °的弯曲。DNA酶I足迹法实验表明，IHF保护一个51 bp的区域，其中包含两个重叠的IHF结合的共识基序。用P-virB-lacZ融合体进行的VirB表达实验表明，在B.在不同培养基中，牛流产杆菌IHF参与了P-virB在细胞内和营养生长过程中的活性调节。一个突变株与20 bp的IHF结合位点置换未能打开virB操纵子在巨噬细胞感染的初始阶段，并显示严重的细胞内增殖缺陷。这些数据表明，IHF在细胞内virB操纵子表达所需的内质网衍生的复制空泡的生物发生过程中起着关键作用。
Type IV secretion systems (T4SSs) are multicomponent machineries that play an essential role in pathogenicity of many facultative intracellular bacteria. The virB operon of Brucella abortus codes for a T4SS essential for virulence and intracellular multiplication. Here, virB expression analyses carried out using lacZ transcriptional fusions showed that virB promoter (P-virB) is temporally activated within J774 cells. Primer extension experiments revealed that virB transcription starts at 27 bp upstream of the first gene of the virB operon. Structural analyses showed that P-virB and regulatory sequences involved in intracellular regulation span 430 bp upstream of the transcription start site. A protein able to bind P-virB was isolated and identified. This protein, homologue to integration host factor (IHF), specifically interacts with P-virB and induces a DNA bending with an angle of 50.36degrees. DNAse I footprinting experiments showed that IHF protects a 51 bp region that contains two overlapped IHF binding consensus motifs. VirB expression experiments carried out with P-virB-lacZ fusions showed that in B. abortus IHF participates in the regulation of P-virB activity during the intracellular and vegetative growth in different media. A mutant strain with a 20 bp IHF binding site replacement failed to turn on the virB operon during the initial stages of macrophage infection and displayed severe intracellular multiplication defects. These data indicate that IHF plays a key role during intracellular virB operon expression being required for the biogenesis of the endoplasmic reticulum-derived replicative vacuole.