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. 2009 Nov 3;106(44):18792-7.
doi: 10.1073/pnas.0900589106. Epub 2009 Oct 21.

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

M Sloan Siegrist  1 , Meera Unnikrishnan, Matthew J McConnell, Mark Borowsky, Tan-Yun Cheng, Noman Siddiqi, Sarah M Fortune, D Branch Moody, Eric J Rubin
Affiliations

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

M Sloan Siegrist et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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.
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.
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.
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.
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).
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References

    1. DiGiuseppe Champion PA, Cox JS. Protein secretion systems in Mycobacteria. Cell Microbiol. 2007;9:1376–1384. - PubMed
    1. Abdallah AM, et al. Type VII secretion—Mycobacteria show the way. Nat Rev Microbiol. 2007;5:883–891. - PubMed
    1. Cole ST, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature. 1998;393:537–544. - PubMed
    1. Tekaia F, et al. Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber Lung Dis. 1999;79:329–342. - PubMed
    1. Gey Van Pittius NC, et al. The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria. Genome Biol. 2001;2:RESEARCH0044. - PMC - PubMed

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