Limits...
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 deletion depletes phosphatidylinositol during iron starvation.(A) Phosphatidylinositol C35:0, neutral mass 852.5728, was detected in the positive mode (B) as [M+H]+ (m/z 853.5802) from triplicate total lipid extracts that were normalized for mass. Chromatogram is representative of triplicate runs. (C) Collision of phosphatidylinositol C35:0 in the negative mode as the [M-H]- ion, m/z 851.5655, showed the expected fragments confirming its structure.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4376628&req=5

ppat.1004792.g005: mbtK deletion depletes phosphatidylinositol during iron starvation.(A) Phosphatidylinositol C35:0, neutral mass 852.5728, was detected in the positive mode (B) as [M+H]+ (m/z 853.5802) from triplicate total lipid extracts that were normalized for mass. Chromatogram is representative of triplicate runs. (C) Collision of phosphatidylinositol C35:0 in the negative mode as the [M-H]- ion, m/z 851.5655, showed the expected fragments confirming its structure.

Mentions: This organism-wide lipidomic search returned a remarkably consistent pattern of significantly decreased signals corresponding to cytoplasmic membrane phospholipids in ΔmbtK, as compared to similarly grown wild type or genetically or chemically complemented mutants. Analysis of molecular feature, m/z 853.5802, illustrates the discovery process. This event matches the known mass of the phosphatidylinositol (PI) ion [M+H]+, C44H85O13P, containing 35 carbon atoms and no unsaturations (C35:0) in its two alkyl chains (Fig. 5A). This event has average intensity counts of 2.1 x 106 in iron-starved wild type, 1.9 x 106 in iron-supplemented wild type, 0.3 x 106 in iron-starved ΔmbtK, 2.2 x 106 in iron-supplemented ΔmbtK, and 2.3 x 106 in iron-starved ΔmbtK complement. Therefore, it meets the criteria for high fold-change and intensity rescue by iron supplementation and complementation. The changes in intensity of PI C35:0 across the five conditions were compared to events corresponding to other PI acyl forms and there were no nearly co-eluting molecules that matched alternatively acylated PI forms. The intensities of these events showed similar patterns among the five conditions that track in parallel with one another and with PI C35:0. The pattern of relative abundance of PI acylforms in wild type and ΔmbtK was similar, suggesting that absence of MbtK did not simply induce a shift in abundance from one acylform to another (S1 Table). Further, key aspects of this computerized analysis were validated in manual analysis of ion chromatograms, which were consistent with a single molecule at the expected retention time for phosphatidylinositol C35:0 (Fig. 5B). CID-MS of m/z 851.5655 was consistent with the [M-H]- ion of phosphatidylinositol C35:0 (Fig. 5C). Although phospholipid concentration is not measured directly in this high throughput method, the intensity count values for time-of-flight MS detection reliably correlate with mass input over a broad concentration range of mycobacterial lipids [28]. Collectively, these data show that the approximately ten-fold change in PI intensity results from mbtK deletion in the setting of iron starvation.


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 deletion depletes phosphatidylinositol during iron starvation.(A) Phosphatidylinositol C35:0, neutral mass 852.5728, was detected in the positive mode (B) as [M+H]+ (m/z 853.5802) from triplicate total lipid extracts that were normalized for mass. Chromatogram is representative of triplicate runs. (C) Collision of phosphatidylinositol C35:0 in the negative mode as the [M-H]- ion, m/z 851.5655, showed the expected fragments confirming its structure.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004792.g005: mbtK deletion depletes phosphatidylinositol during iron starvation.(A) Phosphatidylinositol C35:0, neutral mass 852.5728, was detected in the positive mode (B) as [M+H]+ (m/z 853.5802) from triplicate total lipid extracts that were normalized for mass. Chromatogram is representative of triplicate runs. (C) Collision of phosphatidylinositol C35:0 in the negative mode as the [M-H]- ion, m/z 851.5655, showed the expected fragments confirming its structure.
Mentions: This organism-wide lipidomic search returned a remarkably consistent pattern of significantly decreased signals corresponding to cytoplasmic membrane phospholipids in ΔmbtK, as compared to similarly grown wild type or genetically or chemically complemented mutants. Analysis of molecular feature, m/z 853.5802, illustrates the discovery process. This event matches the known mass of the phosphatidylinositol (PI) ion [M+H]+, C44H85O13P, containing 35 carbon atoms and no unsaturations (C35:0) in its two alkyl chains (Fig. 5A). This event has average intensity counts of 2.1 x 106 in iron-starved wild type, 1.9 x 106 in iron-supplemented wild type, 0.3 x 106 in iron-starved ΔmbtK, 2.2 x 106 in iron-supplemented ΔmbtK, and 2.3 x 106 in iron-starved ΔmbtK complement. Therefore, it meets the criteria for high fold-change and intensity rescue by iron supplementation and complementation. The changes in intensity of PI C35:0 across the five conditions were compared to events corresponding to other PI acyl forms and there were no nearly co-eluting molecules that matched alternatively acylated PI forms. The intensities of these events showed similar patterns among the five conditions that track in parallel with one another and with PI C35:0. The pattern of relative abundance of PI acylforms in wild type and ΔmbtK was similar, suggesting that absence of MbtK did not simply induce a shift in abundance from one acylform to another (S1 Table). Further, key aspects of this computerized analysis were validated in manual analysis of ion chromatograms, which were consistent with a single molecule at the expected retention time for phosphatidylinositol C35:0 (Fig. 5B). CID-MS of m/z 851.5655 was consistent with the [M-H]- ion of phosphatidylinositol C35:0 (Fig. 5C). Although phospholipid concentration is not measured directly in this high throughput method, the intensity count values for time-of-flight MS detection reliably correlate with mass input over a broad concentration range of mycobacterial lipids [28]. Collectively, these data show that the approximately ten-fold change in PI intensity results from mbtK deletion in the setting of iron starvation.

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