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Discovery and molecular investigation of mycobacterial transporters responsible for iron acquisition

Periodic Reporting for period 3 - MycoRailway (Discovery and molecular investigation of mycobacterial transporters responsible for iron acquisition)

Reporting period: 2021-04-01 to 2022-09-30

Mycobacterium tuberculosis is a devastating human pathogen, which killed 1.4 million people in 2019. In order to survive and replicate in the phagosome of macrophages where iron levels are kept extremely low, M. tuberculosis relies on the biosynthesis of siderophores called mycobactins, which capture iron with high affinity inside the host cell. Despite their undisputed importance for virulence, little is known about how these siderophores are exported and imported across the two mycobacterial membranes. The aim of this project is to unravel the structure and mechanism of transporter proteins sitting in the inner and outer mycobacterial membrane involved in the export of empty and the import of iron-loaded mycobactins.
Access to iron represents an Achilles heel of M. tuberculosis and basic research that is conducted in this project to understand iron acquisition at the molecular likely will help to generate new generations of urgently needed drugs against this devastating pathogen.
In the first period we focussed on the structural and functional characterization of the ABC transporter IrtAB (Objective 1). IrtAB is an unusual ABC exporter, which plays a major role in the uptake of iron-loaded siderophores in mycobacteria. In addition, it contains a cytoplasmic siderophore interacting domain (SID) responsible for iron reduction and intracellular release. We obtained a 6.7 Å cryo-EM structure of full-length IrtAB and in addition solved high resolution crystal structures of the SID (1.7 Å) and the transporter devoid of the SID (2.7 Å). Together with biochemical and functional in vivo experiments, we were able to show that IrtAB indeed is a siderophore importer. The structure suggested that the SID is placed adequately to reduce membrane bound siderophores, an unexpected finding that was further supported by in vitro experiments. A manuscript reporting our seminal findings was published in Nature in early 2020.
The aim of objective 2 is the identification of novel transporter proteins involved in siderophore shuttling based on Tn-Seq analysis of Mycobacterium smegmatis. In the meantime, two other groups have published similar Tn-Seq studies as the one planned by us. As the main aim of objective 2 was to characterize novel siderophore shuttling proteins at the biochemical and structural level, we now build on the published studies to investigate interesting candidate proteins.
The aim of objective 3 is to engineer human siderocalin in order to bind mycobacterial siderophores with high affinity. This third aim will be tackled in the second half of the project.
This project bridges the disciplines of structural biology (in particular cryo-EM) with protein biochemistry and microbiology using a unique blend of techniques.
Despite the fact that M. tuberculosis is one of the most extensively studied organisms, its complex cell wall as well as technical hurdles to purify and characterize mycobacterial membrane proteins has so far impeded the molecular investigation of siderophore transport. On the other hand, a number of excellent pioneering studies have shed light on genetic and functional aspects of mycobactin-mediated iron acquisition, upon which experimental work of this proposal builds on. The project will deliver unprecedented insights into proteins responsible for siderophore transport across the two mycobacterial membranes. Further, we will engineer binding proteins to interfere with iron acquisition. Hence, the project will shed light on molecular mechanisms of mycobacterial membrane transport, which is a terra incognita and awaits fascinating novel discoveries. Thereby, our research facilitates the development of novel cures to treat globally emerging multidrug resistant strains of M. tuberculosis.
The mycobacterial ABC exporter IrtAB imports and reduces mycobactins.