Abstract
Iron-starved Mycobacterium tuberculosis utilises the carboxymycobactin-mycobactin siderophore machinery to acquire iron. These two siderophores have high affinity for ferric iron and can withdraw the metal ion from insoluble iron hydroxides and iron-binding proteins. We first reported HupB, a multi-functional mycobacterial protein to be associated with iron acquisition in M. tuberculosis. This 28 kDa cell wall protein, up regulated upon iron limitation functions as a transcriptional activator of mycobactin biosynthesis and is essential for the pathogen to survive inside macrophages. The focus of this study is to understand the role of HupB in iron uptake and transport by the carboxmycobactin-mycobactin siderophore machinery in M. tuberculosis. Experimental approaches included radiolabelled iron uptake studies by viable organisms and protein–ligand binding studies using the purified HupB and the two siderophores. Uptake of 55Fe-carboxymycobactin by wild type M. tuberculosis (WT M.tb.H37Rv) and not by the hupB KO mutant (M.tb.ΔhupB) showed that HupB is necessary for the uptake of ferri-carboxymycobactin. Additionally, the radiolabel recovery was high in HupB-incorporated liposomes upon addition of the labelled siderophore. Bioinformatic and experimental studies using spectrofluorimetry, CD analysis and surface plasmon resonance not only confirmed the binding of HupB with ferri-carboxymycobactin and ferri-mycobactin but also with free iron. In conclusion, HupB is established as a ferri- carboxymycobactin receptor and by virtue of its property to bind ferric iron, functions as a transporter of the ferric iron from the extracellular siderophore to mycobactin within the cell envelope.
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taken from 250 to 200 nm. Panels A, B and C show the spectral changes in HupB upon addition of ferri-carboxymycobactin, ferri-mycobactin and desferri-carboxymycobactin respectively. The upward arrow indicates addition of aliquots of ferri-carboxymycobactin and ferri-mycobactin to a maximal concentration of 3 µM (Panels A and B). In Panel C, a maximal concentration of 50 µM of desferri- carboxymycobactin was added to HupB (Panel C). The spectra in grey colour are control data of buffer (solid lines) and buffer + acetonitrile (dashed lines)
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MC, TNK and NK thank the University Grants Commission for providing fellowship during the doctoral study. MS acknowledges the financial assistance by UPE-II program of University of Hyderabad. KD acknowledges the financial assistance for doctoral studies by the University of Hyderabad.
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Choudhury, M., Koduru, T.N., Kumar, N. et al. Iron uptake and transport by the carboxymycobactin-mycobactin siderophore machinery of Mycobacterium tuberculosis is dependent on the iron-regulated protein HupB. Biometals 34, 511–528 (2021). https://doi.org/10.1007/s10534-021-00292-2
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DOI: https://doi.org/10.1007/s10534-021-00292-2