The Mycobacterium tuberculosis High-Affinity Iron Importer, IrtA, Contains an FAD-Binding Domain

Abstract

Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by Mycobacterium tuberculosis, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of M. tuberculosis in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in M. tuberculosis iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.Mycobacterium tuberculosis, the causative agent of human tuberculosis, like most organisms, requires iron to sustain essential cellular functions. Due to the poor aqueous solubility of the ferric ion (Fe 3ϩ ) in aerobic and neutral pH conditions, free ferric iron is not found in the mammalian host but is bound to iron-binding proteins such as transferrin, lactoferrin, and ferritin (30). A common mechanism by which bacteria acquire iron is the synthesis and secretion of siderophores (high-affinity iron chelators) that can solubilize iron in the environment or remove it from iron-binding proteins of the mammalian host. Fe 3ϩ -siderophore complexes are recognized by specific surface receptors and translocated through the plasma membrane by ABC-type transporters, using the energy generated by ATP hydrolysis (13). Dissociation of iron from the incorporated siderophore complex can occur via cleavage of the siderophore or by the action of a ferric reductase (13). Reduction of Fe 3ϩ results in a weaker binding of Fe 2ϩ to the siderophore, allowing release of iron that can then be utilized (21).To overcome iron limitation, M. tuberculosis synthesizes siderophores named mycobactin and exomycobactin. Mycobactin is very hydrophobic and remains cell associated, whereas exomycobactin (ExMB, also known as carboxymycobactin) is more hydrophilic and is secreted to the medium (8, 16). Fe 3ϩ -ExMB complexes can deliver iron to the cell by transfer of iron to mycobactin (7) or by a pathway that is mycobactin independent (17). Previously, we showed that inactivation of M. tuberculosis irtA (Rv1348) or irtB (Rv1349) genes, which encode membrane proteins of the ABC transporter family (2), results in decreased ability of M. tuberculosis to replicate in low-iron medium and to utilize Fe 3ϩ -ExMb as the sole iron source. Because IrtA and IrtB each encode a membrane protein with one permease domain fused to an ATPase domain, and irtA and irtB are organized in an operon, we postulated that these two proteins associate to form one ABC transporter necessary for iron acquisition in vitro and...

A Ferritin Mutant of Mycobacterium tuberculosis Is Highly Susceptible to Killing by Antibiotics and Is Unable To Establish a Chronic Infection in Mice

“…Shown are mean values (Ϯ standard deviations) from three experiments. grin compared to wild-type strains (29,41). The streptonigrin MIC for the ⌬bfrB strain in 7H9 medium was 1.2 g/ml, whereas the MIC for the wild-type and complemented strains was 10 g/ ml, showing that the level of sensitivity to streptonigrin was highly elevated in the ⌬bfrB strain (Fig.…”

The rv1184c Locus Encodes Chp2, an Acyltransferase in Mycobacterium tuberculosis Polyacyltrehalose Lipid Biosynthesis

“…To determine whether the C-terminal catalytic domain is located in the cytosol or the periplasm, Chp2 was also expressed in M. smegmatis as a theophylline-inducible C-terminal fusion to either alkaline phosphatase (AP) or ␤-galactosidase (␤Gal), as has been performed previously to determine mycobacterial protein topology (Fig. 5C) (36)(37)(38). No AP activity was detected when Chp2 was expressed as the full-length protein, the N terminus (N-term), or the catalytic domain (cat) (Fig.…”

Self-poisoning of Mycobacterium tuberculosis by interrupting siderophore recycling

“…The ATP-binding cassette iron-regulated transporter IrtAB is involved in the uptake of cMBT across the inner membrane (29). Furthermore, as the N-terminal domain of IrtA binds flavin adenine dinucleotide (30), it was suggested that the iron in Fe-cMBT is reduced, as it is imported into the cytoplasm. This observation is consistent with the recycling of cMBTs, which would require a reductive iron release mechanism (1), rather than enzymatic degradation of the iron-loaded siderophores (5).…”

“…and cMBT (Fe-cMBT) are acquired by Mtb by a process dependent on the ESX-3 secretion system (18, 47) (omitted for clarity) likely involving a siderophore receptor. Iron is dissociated from cMBT by IrtAB concomitant with its import (29,30). Iron is dissociated from MBT by a reductive process (31) at a yet-to-be-determined subcellular location.…”