Targeting iron acquisition by Mycobacterium tuberculosis.

Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis continues to be a major worldwide health problem. Lack of compliance to the complex, multi-drug therapy regimen has resulted in multidrug-resistant TB and a need for new drug targets. Siderophore molecules used for iron acquisition are good targets because pathogen survival and virulence is directly related to iron availability. Indeed, a key host defense mechanism is the production of siderocalins that sequester iron-laden siderophores and M. tuberculosis replicates poorly in the absence of these siderophores. A number of investigators have recently targeted siderophores or their synthesis for the development of novel anti-tubercular therapeutics. For example, one group has synthesized 'dominant negative' mycobactin siderophore analogues that significantly inhibit bacterial growth. Several other groups have developed agents that directly inhibit enzymes involved in siderophore synthesis. A profoundly different approach is to target the iron dependent regulator protein (IdeR) that represses siderophore synthesis genes and virulence factors when sustainable iron levels have been achieved. Loss of the repression leads to iron overload and oxidative damage. In contrast, enhanced IdeR repression at low iron levels attenuates M. tuberculosis virulence in mice. The structural basis for iron activation and IdeR binding to DNA has been recently reported and these insights have enabled the structure-based design of agents that target IdeR function. Small peptides that either enhance IdeR repression or inhibit IdeR dimerization demonstrate that IdeR activity can be rationally modulated.

Functional studies of the Mycobacterium tuberculosis iron-dependent regulator.

The iron-dependent regulator (IdeR) protein in Mycobacterium tuberculosis, and its better characterized homologue, the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae, are iron-dependent regulatory proteins that control gene expression in response to iron availability in bacteria. IdeR regulates several genes required for iron uptake and storage including those involved in the synthesis of transition metal chelators called siderophores that are linked to the M. tuberculosis virulence. In this study, the metal ion and binding affinities for IdeR binding to an fxbA operator duplex DNA were estimated using fluorescence assays. The Fe(2+), Co(2+), and Ni(2+) affinities of the two metal ion binding sites in IdeR that are involved in the activation of the regulator DNA binding process in vitro were independently estimated. Binding to the two metal ion binding sites is apparently cooperative and the two affinities differ significantly. Occupation of the first metal ion binding site causes dimerization of IdeR, and the metal ion affinity is about 4 microM for Ni(2+) and much less for Fe(2+) and Co(2+). Binding of the second metal ion fully activates IdeR for binding to the fxbA operator. The equilibrium metal ion dissociation constants for IdeR-fxbA operator binding are approximately 9 microM for Fe(2+), 13 microM for Ni(2+), and 23 microM for Co(2+). Interestingly, the natural IdeR cofactor, Fe(2+), shows high affinities toward both binding sites. These results provide insight into the possible roles for each metal binding site in IdeR activation.