Abstract:
A quarter of the world’s population is infected with latent Mycobacteria tuberculosis. Whilst a latent infection is not fatal, 5-10% of those with latent infections will develop an active, potentially deadly infection during their lifetime. Iron availability is implicated in both latency and reactivation of tuberculosis infections; low iron availability leads to a dormant state which is reversed by increased iron availability. Whilst iron acquisition and storage by Mtb under low iron conditions is well understood, little is known about how iron is released from storage proteins and trafficked to target proteins.
Under iron starvation conditions, Mtb exhibits a transcriptional shift as it adjusts to iron deficiency, downregulating its metabolism and respiration. Furthermore, the major Fe-S cluster biogenesis system, SUF, is prioritised over heme biosynthesis, suggesting that this pathway is important under these conditions. How iron is delivered to the SUF system has yet to be determined. However, there is some evidence that SufT may function as an iron donor to this system. Furthermore, given the importance of iron in Mtb lifestyle, the proteins involved in its mobilisation and trafficking are likely upregulated under iron starvation conditions. While most iron-binding proteins are downregulated, a small collection are significantly upregulated. From this list, we selected three proteins that we propose may be involved in iron mobilisation and trafficking: FprB, a putative ferredoxin-ferredoxin reductase, and RubA and RubB, two putative rubredoxins.
We propose a mechanism in which FprB interacts with iron storage proteins to reduce and release iron, RubA and/or RubB chelate this iron and then interact with SufT to deliver iron to the SUF system. To investigate this hypothesis, purification protocols were developed for all proteins, except Mtb FprB, which was largely insoluble. The potential interactions between RubA, RubB and SufT were investigated using native-PAGE and size-exclusion chromatography. These experiments indicate that in the presence of Fe(II), RubA and SufT interact and that SufT may form a large homomultimer. In the context of the proposed mechanism, these results indicate that RubA may chelate iron and interact with SufT. Future investigations into this proposed mechanism are made possible with the protocols developed in this project.