The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages

Abstract

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron‐dependent regulator), an iron‐responsive DNA‐binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB‐like) and others annotated to be involved in the synthesis of iron‐storage proteins (bfrA, bfrB). Some putative IdeR‐regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)‐like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA–mbtB, mbI, rv3402c and bfrA–bfd, for interaction with IdeR and for iron‐dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the −10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14‐ to 49‐fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR‐ and iron‐repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP‐1 macrophages.

Nonsteroidal anti-inflammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials

“…NMR spectra for 4-OH-OPB and its ringopened quinonimine were consistent with those described by Dekkers et al (24). The addition of a hydroxyl to the pyrazolidinedione ring was consistent with a large 13 C shift of the C4 carbon from 42 ppm (seen in OPB) to 81 ppm (seen in 4-OH-OPB) using 2D 13 C-heteronuclear single-quantum coherence (HSQC) and 2D 13 C-heteronuclear multiple-bond correlation (HMBC) spectra. Bioactivity of 4-OH-OPB.…”

“…The time course was initiated on addition of an aliquot of 1 M NaNO 2 in D 2 O to give a final NaNO 2 concentration 10-fold higher than the sample concentration. The 1D 1 H spectra were collected at intervals for up to 18 h. Natural abundance 13 C chemical shifts were obtained at 298 K using 2D 13 C-HSQC (51) and 2D 13 C-HMBC (52) spectra. Natural abundance data were collected at sample concentrations from 25 to 100 mM using 25%, 58%, and 75% d 6 -DMSO solutions mixed with PBS prepared in D 2 O at pH 5.0.…”

“…Mtb can occupy diverse microenvironments in the host. Evidence from auxotrophs, analyses of gene expression, and direct and indirect biochemical measurements in vivo or ex vivo in experimental animals and people suggest that such environments expose Mtb to acid, hypoxia, reactive nitrogen intermediates (RNIs), oxidative stress, carbohydrate deficiency, and metal starvation or intoxication, and require Mtb to metabolize fatty acids or cholesterol (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In vitro, many of the same conditions can make Mtb relatively refractory to killing by the standard agents, except for pyrazinamide, which is only effective at a low pH.…”

Transcriptional regulation of Mycobacterium tuberculosis hupB gene expression

“…The binding of IdeR to hupB promoter DNA was demonstrated by EMSA according to Gold et al (2001) with minor modifications. The 340 bp hupB promoter DNA, encompassing 2235 to +105 bp with respect to the start point of hupB, was PCR-amplified (forward: 59-CATATGCG-CACGCACAATCG-39/reverse: 59-AAGCTTCGCGCACTCTACCC-39) as described above.…”

“…The DNA footprinting assay was performed according to Gold et al (2001). This was done using the 340 bp hupB promoter DNA and the two PCR-amplified 189 and 171 bp products, respectively.…”