Characterization of Periplasmic Iron Transport in Mycobacterium tuberculosis

Abstract

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis (Mtb), results in 10 million infections and 1.5 million deaths annually. Current TB treatments are typically a cocktail of up to five antibiotics that needs to be administered for a 9‐month duration; unfortunately, several of these drugs illicit severe side effects. These long, harsh treatments lead to patient non‐compliance resulting in a rise in multiple‐drug resistant Mtb strains ‐ MDR‐TB. Due to these factors new treatment strategies are needed. As host iron acquisition is essential for Mtb’s survival, elucidating key players in the Mtb iron uptake pathways may yield good drug targets. Mtb predominately acquires host iron by the siderophore‐mediated uptake pathway, in which small molecules with high affinity for iron are secreted to scavenge for host iron. We seek to shed light on the mechanism by which ferric‐siderophores are transported into the Mtb cytosol. At present there is little known about the proteins required to shuttle ferric‐siderophores across the outer membrane, cell‐wall environment, and periplasmic space to the inner membrane. There are two putative Mtb periplasmic binding proteins (PBPs), FecB and FecB2, and we hypothesize that one or both of these PBPs shuttle ferric‐siderophores across the periplasmic space. To test this, we examined the affinity of FecB and FecB2 for the Mtb secreted ferric‐siderophore, ferric‐carboxymycobactin (Fe‐cMB), by tryptophan fluorescence quenching titration assays. The affinity of FecB for Fe‐cMB was in the high nanomolar range (Kd = 355 ± 146 nM); in contrast, the affinity of FecB2 was in the high micromolar range. This result suggests that FecB is involved in transporting Fe‐cMB across the periplasmic space. To further probe the FecB residues involved in Fe‐cMB binding, we carried out a comprehensive mutational analysis. In an attempt to decipher the protein‐protein interaction network of FecB, we utilized co‐immunopreciptation (co‐IP) of FLAG‐tagged FecB in the non‐pathogenic Mtb model organism Mycobacterium smegmatis, followed by mass spectrometry analysis. The co‐IP FLAG‐FecB experiments resulted in identification of known proteins in the import of Fe‐cMB and surprisingly, also proteins thought to be involved in the export of apo‐siderophores. Together, these data clearly place FecB in the siderophore‐mediated iron‐uptake pathway, and will be discussed in more detail. Finally, this work has broadened our understanding of the Mtb iron acquisition pathway, which may lead to the identification of new therapeutic targets.