Magnesium depletion triggers production of an immune modulating diterpenoid in Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) is the causative agent of the human disease Tuberculosis, and remains a worldwide health threat responsible for ∼1.7 million deaths annually. During infection, Mtb prevents acidification of the engulfing phagosome, thus blocking endocytic progression and eventually leading to stable residence. The diterpenoid metabolite isotuberculosinol (isoTb) exhibits biological activity indicative of a role in this early arrest of phagosome maturation. Presumably, isoTb production should be induced by phagosomal entry. However, the relevant enzymatic genes are not transcriptionally upregulated during engulfment. Previous examination of the initial biosynthetic enzyme (Rv3377c/MtHPS) involved in isoTb biosynthesis revealed striking inhibition by its Mg(2+) cofactor, leading to the hypothesis that the depletion of Mg(2+) observed upon phagosomal engulfment may act to trigger isoTb biosynthesis. While Mtb is typically grown in relatively high levels of Mg(2+) (0.43 mM), shifting Mtb to media with phagosomal levels (0.1 mM) led to a significant (∼10-fold) increase in accumulation of the MtHPS product, halimadienyl diphosphate, as well as easily detectable amounts of the derived bioactive isoTb. These results demonstrate isoTb production by Mtb specifically under conditions that mimic phagosomal cation concentrations, and further support a role for isoTb in the Mtb infection process.

Figure 1. Isotuberculosinol biosynthesis

Synthesis of isotuberculosinol proceeds via the cyclization of geranylgeranyl diphosphate (GGPP) to halimadienyl diphosphate (HPP) via the MtHPS encoded by Rv3377c, which is further transformed to isotuberculosinol (isoTb) via the enzyme encoded by Rv3378c.

Figure 2. Magnesium depletion triggers increased HPP production by MtHPS in E. coli

Production of diterpenoid metabolites in E. coli expressing a GGPP synthase and MtHPS. Legend: High Mg²⁺ (0.43 mM) (open circles), Low Mg²⁺ (filled circles). Shown is the average, with error bars indicating the standard deviation, of two independent biological samples.

1. Concentration of GGPP per gram of bacterial fresh weight over time.

2. Concentration of HPP per gram of bacterial fresh weight over time.

Figure 3. Biosynthesis of isoTb is triggered by Mg²⁺ depletion

Selected ion (m/z = 119 + 191 + 290) chromatogram from GC-MS analysis of extracted diterpenoid metabolites from M. tuberculosis H37Rv grown in 7H9 media and transferred into new 7H9 media containing either the normal high (0.43 mM) or low (0.1 mM) concentration of Mg²⁺ compared to isoTb standard reference chromatogram (as indicated). The chromatograms are vertically offset for clarity, but those of the extracts from high and low [Mg²⁺] exposed Mtb cultures are shown on the same absolute scale.

Figure 4. Magnesium depletion triggers diterpenoid metabolism in Mtb

Mtb CDC1551 grown in 7H9 media for 4 weeks (“7H9”) or grown in 7H9 for 2 weeks and new 7H9 media for 2 weeks (“7H9→7H9”) show distinctly different production profiles of diterpenoid metabolites HPP (detected here as the derived HaOH) and IsoTb from bacteria subject to growth in 7H9 and subsequent shift to 7H9 containing 0.1 mM Mg²⁺ and 2.5 mM Mn²⁺ (“7H9→Low”). However, the change in environmental media conditions does not appear to effect availability of the precursor molecule, GGPP (detected here as the derived GGOH). (N.D. = not detected, L.D. = concentrations at or below limit of positive identification, 20 ng/L culture). Shown is the average, with error bars indicating the standard deviation, of two independent biological samples.

Figure 5. Proposed regulation of isotuberculosinol biosynthesis during phagosomal engulfment

During infection, Mtb (purple) is compartmentalized within the phagosome (light grey) of the macrophage (dark grey). IsoTb is known to interfere with early stages of phagosomal maturation, which prevents later lysosomal (greyscale) fusion and bactericidal activity. IsoTb biosynthesis is inhibited by high Mg²⁺ concentration until phagosomal engulfment, when the bacteria are subjected to a shift to a low Mg²⁺ environment, releasing inhibition of the committed step of isoTb biosynthesis.