Modification of 4-Fold and B-Pores in Bacterioferritin from Mycobacterium tuberculosis Reveals Their Role in Fe²⁺ Entry and Oxidoreductase Activity

Abstract

The self-assembled ferritin nanocages, nature's solution to iron toxicity and its low solubility, scavenge free iron to synthesize hydrated ferric oxyhydroxide mineral inside their central cavity by protein-mediated ferroxidase and hydrolytic/nucleation reactions. These complex processes in ferritin commence with the rapid influx of Fe 2+ ions via the inter-subunit contact points (i.e., pores/channels). Investigation of these pores as Fe 2+ uptake routes in ferritins remains a subject of intense research, in iron metabolism, toxicity, and bacterial pathogenesis, which are yet to be established in the bacterioferritin (BfrA) from Mycobacterium tuberculosis (Mtb). The electrostatic properties of this protein indicate that the 4-fold and B-pores might serve as potential Fe 2+ entry routes. Therefore, in the current work, electrostatics at/along these pores was altered by site-directed mutagenesis to establish their role in Fe 2+ uptake/oxidation (ferroxidase activity) in Mtb BfrA. Despite forming self-assembled protein nanocompartment, these 4-fold and B-pore variants exhibited partial loss of ferroxidase activity and lower accumulation of transient species, which not only indicated their role in Fe 2+ entry but also suggested the existence of multiple pathways. Although the B-pore variants inhibited the rapid ferroxidase activity to a larger extent, they had minimal impact on their cage stability. The current work revealed the relative contribution of these pores toward rapid Fe 2+ uptake/oxidation and cage stability, possibly as consequences of their differential symmetry, number of modified residues (at each pore), and heme content. Therefore, these findings may help to understand the role of these pores in iron acquisition and Mtb proliferation under iron-limiting conditions to control its pathogenesis.

Serum Ceruloplasmin-to-Albumin Ratio as a Biochemical Marker in Pulmonary Tuberculosis Before and After Treatment

“…The serum ceruloplasmin-to-albumin ratio in newly diagnosed TB patients was 0.022 ± 0.003, decreasing to 0.008 ± 0.001 post-treatment. This decline supports the utility of these biomarkers in assessing disease severity and treatment response [13][14][15]. Our research underscores the potential of serum biomarkers not only in diagnosing PTB but also in monitoring treatment efficacy.…”

Targeting Siderophore-Mediated Iron Uptake in M. abscessus: A New Strategy to Limit the Virulence of Non-Tuberculous Mycobacteria

“…In mycobacterial infections, the survival of the bacilli in the host relies on the efficient uptake of this metal, which acts as a co-factor in several biological processes. Therefore, there is a growing interest in targeting the pathways involved in its acquisition [ 34 , 35 , 36 ]. In detail, the production of siderophores (mycobactins and carboxymycobactins) has been shown to be of crucial importance; hence, proteins involved in their assembly, transport, and functionality have been proposed as potential targets [ 37 , 38 , 39 , 40 , 41 , 42 ].…”

Advancements of the Molecular Directed Design and Structure–Activity Relationship of Ferritin Nanocage

“…Among them, the 3-fold axis channel of animal ferritin is considered the main entry pathway for iron, and the 4-fold axis channel and 3-fold axis hydrophilic channels of plant ferritin serve as pathways for iron diffusion into its lumen . In addition, the dimer meeting the other side-on forms 24 asymmetric channels, called B-pores, which are the main entry and exit channels for Fe atoms in bacterial and diatom ferritin. − …”