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Lipidomic analysis links mycobactin synthase K to iron uptake and virulence in M. tuberculosis.

Madigan CA, Martinot AJ, Wei JR, Madduri A, Cheng TY, Young DC, Layre E, Murry JP, Rubin EJ, Moody DB - PLoS Pathog. (2015)

Bottom Line: The mbtK mutant showed markedly reduced iron scavenging and growth in vitro.The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids.Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.

No MeSH data available.


Related in: MedlinePlus

mbtK is required for growth during iron-starvation and early virulence in vivo.(A) Liquid M. tb cultures grown in iron-depleted medium, supplemented or not with 50 μM ferric chloride. (B) Representative plates from three replicates of the strains shown in (A) grown for 3 weeks on iron-depleted plates supplemented or not with 50 μM ferric chloride. (C) ΔmbtK and complemented ΔmbtK, marked chromosomally with unique identifiers (q-tags), were mixed 50:50 and used to infect fifteen C57/B6 mice at ~1,000 CFU via aerosol. Five mice were sacrificed at each time point. Lung homogenates were plated for CFU, colonies were counted and collected from plates to prepare genomic DNA. Average total recovered CFU were 1,131, 68,355 and 655,650 at 24 hours, 1 week and 6 weeks, respectively. Quantitative PCR for the q-tag specific to each strain was performed in duplicate, resulting in chromosomal equivalents (CEQ) of each strain to the total CEQ recovered per lung [37]. Log ratios were evaluated by unpaired T-tests.
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ppat.1004792.g004: mbtK is required for growth during iron-starvation and early virulence in vivo.(A) Liquid M. tb cultures grown in iron-depleted medium, supplemented or not with 50 μM ferric chloride. (B) Representative plates from three replicates of the strains shown in (A) grown for 3 weeks on iron-depleted plates supplemented or not with 50 μM ferric chloride. (C) ΔmbtK and complemented ΔmbtK, marked chromosomally with unique identifiers (q-tags), were mixed 50:50 and used to infect fifteen C57/B6 mice at ~1,000 CFU via aerosol. Five mice were sacrificed at each time point. Lung homogenates were plated for CFU, colonies were counted and collected from plates to prepare genomic DNA. Average total recovered CFU were 1,131, 68,355 and 655,650 at 24 hours, 1 week and 6 weeks, respectively. Quantitative PCR for the q-tag specific to each strain was performed in duplicate, resulting in chromosomal equivalents (CEQ) of each strain to the total CEQ recovered per lung [37]. Log ratios were evaluated by unpaired T-tests.

Mentions: Although MbtN is necessary for synthesis of unsaturated siderophores, the role, if any, of the unsaturation in iron scavenging and growth is unknown. For M. tb H37Rv ΔmbtK, we expected the absence of mycobactin to attenuate growth in vitro when heme iron was unavailable; however, production of biologically active unacylated mycobactin peptides or passive iron uptake might allow some iron scavenging. We grew ΔmbtK and ΔmbtN starter cultures in iron-depleted medium supplemented with 50 μM ferric chloride, washed them in iron-depleted medium, divided cultures in half, and inoculated each half into medium that was supplemented or not with iron. Whereas wild type and ΔmbtN grew well in iron-depleted medium, ΔmbtK was entirely unable to grow over eight days (Fig. 4A). Supplementation with 50 μM ferric chloride substantially rescued ΔmbtK growth, although a growth lag of two days was observed. Thus, ΔmbtK is essential for growth in vitro and the defect relates specifically to impaired iron scavenging. To confirm these results on solid medium, we plated the strains in triplicate on iron-depleted or iron-supplemented agar plates, and again observed scant growth of ΔmbtK on iron-depleted medium. On iron-supplemented agar medium, the relative growth defect of ΔmbtK was partially reversed (Fig. 4B). Thus, MbtK, but not MbtN, is required for non-heme iron acquisition and iron-dependent growth in vitro.


Lipidomic analysis links mycobactin synthase K to iron uptake and virulence in M. tuberculosis.

Madigan CA, Martinot AJ, Wei JR, Madduri A, Cheng TY, Young DC, Layre E, Murry JP, Rubin EJ, Moody DB - PLoS Pathog. (2015)

mbtK is required for growth during iron-starvation and early virulence in vivo.(A) Liquid M. tb cultures grown in iron-depleted medium, supplemented or not with 50 μM ferric chloride. (B) Representative plates from three replicates of the strains shown in (A) grown for 3 weeks on iron-depleted plates supplemented or not with 50 μM ferric chloride. (C) ΔmbtK and complemented ΔmbtK, marked chromosomally with unique identifiers (q-tags), were mixed 50:50 and used to infect fifteen C57/B6 mice at ~1,000 CFU via aerosol. Five mice were sacrificed at each time point. Lung homogenates were plated for CFU, colonies were counted and collected from plates to prepare genomic DNA. Average total recovered CFU were 1,131, 68,355 and 655,650 at 24 hours, 1 week and 6 weeks, respectively. Quantitative PCR for the q-tag specific to each strain was performed in duplicate, resulting in chromosomal equivalents (CEQ) of each strain to the total CEQ recovered per lung [37]. Log ratios were evaluated by unpaired T-tests.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376628&req=5

ppat.1004792.g004: mbtK is required for growth during iron-starvation and early virulence in vivo.(A) Liquid M. tb cultures grown in iron-depleted medium, supplemented or not with 50 μM ferric chloride. (B) Representative plates from three replicates of the strains shown in (A) grown for 3 weeks on iron-depleted plates supplemented or not with 50 μM ferric chloride. (C) ΔmbtK and complemented ΔmbtK, marked chromosomally with unique identifiers (q-tags), were mixed 50:50 and used to infect fifteen C57/B6 mice at ~1,000 CFU via aerosol. Five mice were sacrificed at each time point. Lung homogenates were plated for CFU, colonies were counted and collected from plates to prepare genomic DNA. Average total recovered CFU were 1,131, 68,355 and 655,650 at 24 hours, 1 week and 6 weeks, respectively. Quantitative PCR for the q-tag specific to each strain was performed in duplicate, resulting in chromosomal equivalents (CEQ) of each strain to the total CEQ recovered per lung [37]. Log ratios were evaluated by unpaired T-tests.
Mentions: Although MbtN is necessary for synthesis of unsaturated siderophores, the role, if any, of the unsaturation in iron scavenging and growth is unknown. For M. tb H37Rv ΔmbtK, we expected the absence of mycobactin to attenuate growth in vitro when heme iron was unavailable; however, production of biologically active unacylated mycobactin peptides or passive iron uptake might allow some iron scavenging. We grew ΔmbtK and ΔmbtN starter cultures in iron-depleted medium supplemented with 50 μM ferric chloride, washed them in iron-depleted medium, divided cultures in half, and inoculated each half into medium that was supplemented or not with iron. Whereas wild type and ΔmbtN grew well in iron-depleted medium, ΔmbtK was entirely unable to grow over eight days (Fig. 4A). Supplementation with 50 μM ferric chloride substantially rescued ΔmbtK growth, although a growth lag of two days was observed. Thus, ΔmbtK is essential for growth in vitro and the defect relates specifically to impaired iron scavenging. To confirm these results on solid medium, we plated the strains in triplicate on iron-depleted or iron-supplemented agar plates, and again observed scant growth of ΔmbtK on iron-depleted medium. On iron-supplemented agar medium, the relative growth defect of ΔmbtK was partially reversed (Fig. 4B). Thus, MbtK, but not MbtN, is required for non-heme iron acquisition and iron-dependent growth in vitro.

Bottom Line: The mbtK mutant showed markedly reduced iron scavenging and growth in vitro.The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids.Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.

No MeSH data available.


Related in: MedlinePlus