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.

FIG. 1.

Complementation of ST73. H37Rv (⧫), ST73 (•), and ST73 transformed with a plasmid expressing irtA (▴), irtB (▪), or irtAB (×) were inoculated in LIMM. Growth was monitored each day by measuring the OD₅₄₀. The results of a representative experiment are shown. The experiment was repeated three times. All strains grew normally in high-iron medium (data not shown).

FIG. 2.

⁵⁵Fe³⁺-ExMB uptake. H37Rv and ST73 were cultured in LIMM and incubated with ⁵⁵Fe³⁺-ExMB at 0 or 37°C. At the indicated time points, an aliquot of cells was collected, washed as described in Materials and Methods, and resuspended in scintillation liquid. The radioactivity incorporated was measured in a Beckman scintillation counter. Symbols: □, H73Rv; ○, ST73. The data show the radioactivity incorporated at 37°C after subtraction of the radioactivity incorporated at 0°C. The average and standard deviation are derived from three experiments.

FIG. 3.

Sequence analysis of IrtA-NTD. A sequence alignment of the M. tuberculosis IrtA-NTD with other putative iron-siderophore utilization proteins is shown. The sequences were aligned by using CLUSTAL W (25), and the figure was generated with ALSCRIPT (1). Terms: SIP-S.Putr, siderophore-interacting protein from Shewanella putrefaciens; SID-IrtAMtb, IrtA-NTD from M. tuberculosis; MxcB-S.aura, myxochelin iron-binding protein from Stigmatella aurantiaca; PSUP-P.put, putative iron-chelator utilization protein from Pseudomonas putida; ViuB-V.chol, vibriobactin utilization protein from V. cholerae; ViuB-R.bal, vulnibactin utilization protein from Rhodopirellula baltica. The secondary structural elements at the top of the sequences are from the crystal structure of SIP-S.putr (PDB ID 2GPJ). Cylinders represent α-helices, and arrows denote β-strands. Residues labeled with an asterisk at the bottom of the sequences are those in the SIP-S.putr structure whose side chains interact with the bound FAD. These residues are identical among the aligned sequences. Residues marked with a triangle are those having direct hydrogen bonds from main chain atoms to the bound FAD. A vertical arrow marks the domain boundary between the N- and C-terminal domains. The C-terminal domain of these proteins has a β₁α₁β₂α₂β₃α₃ Rossmann fold, α₄ connecting β₃ and β₄, and a second Rossmann fold lacking the sixth strand as is observed in other FAD-binding proteins (4).

FIG. 4.

IrtA-NTD purification. IrtA-NTD was expressed and purified as described in Materials and Methods. The protein was expressed with an N-terminal His₆ tag and migrated as a band of ∼32 kDa on an SDS-polyacrylamide gel (8 to 25%). The calculated molecular mass is 31.6 kDa. After the His tag was cleaved by TEV protease, the calculated molecular mass is 28.5 kDa. Lane M, molecular markers with molecular masses labeled on the left side in kilodaltons.

FIG. 5.

Spectral analysis of IrtA-NTD. (A) UV/visible spectrum of purified IrtA-NTD. An inset shows an expanded-scale spectrum of characteristic flavin absorbance between 300 and 500 nm. (B) UV/visible spectrum of the filtrate recovered after Gnd-HCl denaturation of IrtA-NTD and filtration through a Microcon YM-10 column.

FIG. 6.

Growth of mycobacterial strains in low iron and stability of mutated IrtAs. (A) M. tuberculosis strains were grown in LIMM. Growth was monitored each day by measuring the OD₅₄₀. H37Rv (⧫), ST73 (□), and ST73 complemented with wild-type irtAB (▵), irtAR70A-irtB (▪), irtAY72A-irtB (▴), and irtAT73A-irtB (•) were examined. The results of one representative experiment are shown. The experiment was performed three times. (B) Stability of noncomplementing mutated IrtAs. S-tagged IrtA wild type (lane 1), S-IrtAY72A (lane3) and S-IrtAT73A (lane 4) were expressed in M. smegmatis under acetamide induction. M. smegmatis harboring the acetamide-inducible empty vector was used as a negative control (lane 2). Membrane fractions were isolated, the protein concentration was determined, and an equivalent amount of protein from each extract was loaded on a SDS-7.5% PAGE gel. S-IrtAs were detected by Western analysis with an anti-S monoclonal antibody.

FIG. 7.

Determination of NTD-IrtA membrane topology. The β-galactosidase (A) and alkaline phosphatase (B) activities of M. smegmatis expressing IrtA-NTD fused at various points to LacZ and PhoA were determined. The activities were measured in M. smegmatis, cultured in LIMM as described in Materials and Methods. The averages and standard deviations of three independent experiments are shown. M. smegmatis transformed with the vectors pJM12 or pJM11 was used as negative controls. (C) Anti-PhoA Western analysis of M. smegmatis lysates expressing each IrtA-NTD-PhoA fusion. Lanes: 1, pJM11 control; 2, A129-phoA; 3, V150-phoA; 4, R274-phoA; 5, S320-phoA. (D) Predicted topology of IrtA-NTD based on the LacZ and PhoA activities of translational fusions constructed in the present study (indicated by filled circles) and the position of the first transmembrane domain predicted by HMMTOP (pointed by arrows). Additional transmembrane domains illustrated are based on the prediction by HMMTOP.

FIG. 8.

Model structure showing the FAD-binding pocket IrtA-NTD. The model was constructed based on sequence alignments and the structure of the SUP from S. putrefaciens (PDB entry 2GPJ). Only side chains that are directly involved in the binding interactions are shown. These side chains are conserved in most of the SUP sequences. There are also many H-bonds and pi-electron interactions with the protein main chain, which are not shown in the figure for clarity. The residue numbers are from the IrtA protein sequence. The figure was generated by using the program PYMOL (http://www.pymol.org).

FIG. 9.

Model of IrtA-NTD function. IrtA-NTD is proposed to function as a flavin/ferric reductase that reduces iron in the imported Fe³⁺-ExMB complex for its assimilation.