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Updated: Jun 20, 2026
Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria
Published on: October 31, 2019
Identification and functional characterization of the iron-dependent regulator (IdeR) of Mycobacterium avium subsp.
Harish K Janagama1, T M A Senthilkumar1, John P Bannantine2
1Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN, USA.
Abstract:
Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin-dependent for cultivation in vitro. The iron-dependent regulator (IdeR) is a well-characterized global regulator responsible for maintaining iron homeostasis in Mycobacterium tuberculosis (MTB). We identified an orthologous segment in the MAP genome, MAP2827, with >93 % amino acid identity to MTB IdeR. Electrophoretic mobility shift assays and DNase protection assays confirmed that MAP2827 binds the 19 bp consensus motif (iron box) on the MAP genome. Sequencing of MAP2827 from multiple isolates revealed a non-synonymous change (R91G) exclusive to sheep strains. Reporter gene assays and quantitative real-time RT-PCR assays in two diverse MAP strains and in an ideR deletion mutant of M. smegmatis (mc(2)155) suggested that both sheep MAP IdeR (sIdeR) and cattle MAP IdeR (cIdeR) repress mbtB transcription at high iron concentrations and relieve repression at low iron concentrations. On the other hand, bfrA (an iron storage gene) was upregulated by cIdeR when presented with MTB or the cattle MAP bfrA promoter, and was downregulated by sIdeR in the presence of MTB, or sheep or cattle MAP bfrA promoters, at high iron concentrations. The differential iron regulatory mechanisms between IdeR-regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP.
Insights
Mycobacterium avium subspecies paratuberculosis (MAP) IdeR regulators show strain-specific differences in iron management. These variations in iron acquisition and storage may explain distinct pathogenic traits in cattle and sheep strains.
Area of Science:
- * Microbiology
- * Molecular Biology
- * Animal Health
Background:
- * Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in livestock.
- * MAP requires mycobactin for in vitro cultivation due to unique iron needs.
- * The iron-dependent regulator (IdeR) controls iron homeostasis in Mycobacterium tuberculosis (MTB).
Purpose of the Study:
- * To identify and characterize the IdeR ortholog in MAP.
- * To investigate the role of MAP IdeR in regulating iron homeostasis.
- * To explore potential differences in IdeR function between cattle and sheep MAP strains.
Main Methods:
- * Bioinformatic analysis to identify MAP2827 as the IdeR ortholog.
- * Electrophoretic mobility shift and DNase protection assays to confirm DNA binding.
- * Reporter gene assays and qRT-PCR to assess gene regulation by IdeR.
Main Results:
- * MAP2827 exhibits high identity to MTB IdeR and binds the conserved 'iron box' motif.
- * A specific mutation (R91G) in IdeR was found exclusively in sheep MAP strains.
- * Both cattle and sheep IdeR regulate mbtB transcription differently based on iron levels.
- * Cattle IdeR upregulates bfrA, while sheep IdeR downregulates it, indicating strain-specific iron storage regulation.
Conclusions:
- * Strain-specific IdeR variants in MAP contribute to differential iron regulation.
- * These regulatory differences may underlie distinct growth and pathogenicity in cattle and sheep.
- * The findings offer insights into MAP's mycobactin dependency and iron metabolism.
