Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition

Abstract

The Esx secretion pathway is conserved across Gram-positive bacteria. Esx-1, the best-characterized system, is required for virulence of Mycobacterium tuberculosis, although its precise function during infection remains unclear. Esx-3, a paralogous system present in all mycobacterial species, is required for growth in vitro. Here, we demonstrate that mycobacteria lacking Esx-3 are defective in acquiring iron. To compete for the limited iron available in the host and the environment, these organisms use mycobactin, high-affinity iron-binding molecules. In the absence of Esx-3, mycobacteria synthesize mycobactin but are unable to use the bound iron and are impaired severely for growth during macrophage infection. Mycobacteria thus require a specialized secretion system for acquiring iron from siderophores.

Fig. 1.

Construction of the mycobacterial esx-3 mutants. Schematic representation of the esx-3 loci from Mycobacterium smegmatis (upper) and in Mycobacterium tuberculosis/Mycobacterium bovis bacillus Calmette–Guérin (BCG) (lower) (A) and strategies used to generate the M. bovis BCG esx-3 conditional mutant (BCG-tet-esx-3) (B) and the M. smegmatis Δesx-3 mutant (C).

Fig. 2.

Esx-3 is required for Mycobacterium bovis bacillus Calmette–Guérin (BCG) and Mycobacterium smegmatis growth and for secretion of esx-3-encoded proteins under iron-deprived conditions. Growth of BCG-tet-esx-3 in iron-replete 7H9 medium or low iron glycerol alanine salts Tween-80 medium in the presence or absence of the inducer anhydrotetracycline (n = 3; **, P = 0.0002) (A). Growth of wild-type (WT) and Δesx-3 M. smegmatis in low iron (100 μM 2,2′-dipyridyl) or iron-replete (12.5 μM FeCl₃) Sauton's medium (n = 3; **, P = 0.0014) (B). Strains were subcultured in low iron Sauton's medium before inoculation. Growth of fxbA⁻ and fxbA⁻ Δmsmeg0617 M. smegmatis containing pJEB402 with or without the msmeg0617 gene in low iron Sauton's medium (C). Strains were not subcultured before inoculation. Anti-c-myc and anti-ClpP1P2 immunoblotting of cell lysates and culture supernatants of WT and Δesx-3 M. smegmatis containing pTetG-esxGH-c-myc (D) or WT M. bovis BCG containing pMV762-esxG-6Xhis-esxH-c-myc (E) in low iron or iron-replete Sauton's medium. All data are representative of at least three independent experiments. Error bars represent the standard deviations.

Fig. 3.

Esx-3 interacts with the mycobactin pathway but is not required for sensing iron or for siderophore synthesis. (A) Growth of wild-type (WT), Δesx-3, exochelin (fxbA⁻), and mycobactin (mbtD⁻) insertional biosynthesis mutants and double mutants (fxbA⁻ Δesx-3 and fxbA⁻ mbtD⁻) in low iron or iron-replete Sauton's medium (n = 3; *, P < 0.001). Strains were not subcultured before inoculation. (B) Quantitative RT-PCR for mbtB, mbtL, and irtA expression in WT and Δesx-3 Mycobacterium smegmatis (n = 3) in low iron (LI) or iron-replete (IR) medium and for mbtB in bacillus Calmette–Guérin (BCG)-tet-esx-3 in low iron medium. Data representative of three independent experiments are shown. Error bars represent the standard deviations. (C) The LC-MS analysis of lipids extracted from the whole cell lysates of M. smegmatis with chlorofrom and methanol yielded mycobactin chromatograms in which the m/z (881.4), elution time, and fragments (right) correspond to the expected and actual measurements of an authentic standard. Conditioned supernatants were subjected to LC-MS detection in mass windows corresponding to the known masses of carboxymycobactins [m/z (756.7–757.7) + 14n, where n is 0, 1, 2, 3, 4] and [m/z (757.7–758.7) + 14n] (upper). The elution time and collision-induced dissociation MS fragmentation (right) of the putative carboxymycobactins are consistent with those of an authentic carboxymycobactin standard.

Fig. 4.

Esx-3 is required for optimal utilization of iron-bound mycobactin. Growth curves for fxbA⁻, fxbA⁻ mbtD⁻, and fxbA⁻ Δesx-3 Mycobacterium smegmatis in low iron Sauton's medium (n = 3; **, P = 0.0005) (A), in the presence of 10 ng/mL ferri-mycobactin S (n = 3; **, P = 0.0017) (B), or 20 ng/mL ferri-carboxymycobactin S (n = 3; **, P = 0.0003) (C). All data are representative of at least three independent experiments. Error bars represent the standard deviations.

Fig. 5.

Esx-3 is required for mycobacterial infection of macrophages. Growth of wild-type (WT) M. bovis BCG and BCG-tet-esx-3 in the presence or absence of anhydrotetracycline in J774 macrophages (n = 3).