Deciphering sulfoglycolipids of Mycobacterium tuberculosis

Abstract

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.

Fig. 1.

Purification of Ac₂-, Ac₃-, Ac₄SGL of M. tuberculosis H37Rv. A: Structure of the major M. tuberculosis H37Rv Ac₄SGL is shown. Position (1) is always acylated by a palmitic or a stearic acid, while positions (2), (3), and (4) are acylated mainly by HPA but also by PA. Ac₂SGL are acylated in positions (1) and (2), and Ac₃SGL are acylated in positions (1), (2), and (3). B: Structure of SL-I to SL-IV. C: Purification schemes are shown for the different acyl forms of SGL. D: TLC analysis of the different fractions issued from the silicic column eluted by CHCl₃ (1, 2), CHCl₃/CH₃OH 9/1 (3, 4), CHCl₃/CH₃OH 8/2 (5, 6), CHCl₃/CH₃OH 7/3 (7, 8), and CH₃OH (9). AS, acetone-soluble fraction. F4 to F7 contained SGL. E: TLC analysis of the different acyl forms of SGL after purification. All the TLC were developed with CHCl₃/CH₃OH, 85:15, v/v, and sprayed with anthrone.

Fig. 2.

MALDI MS spectra in negative-ion mode of M. tuberculosis H37Rv Ac₂SGL (A–C), Ac₃SGL (D–F), and Ac₄SGL (G–I). The different views correspond to different expanded areas. In panels B, E, and H, the isotopic profile of one major ion is presented. In panels A, D, and G, the arrows indicate the species targeted for MS/MS analysis shown in Fig. 3 and supplementary Fig. II. In panels C, F, and I, the species containing HPA are indicated by asterisks, the species containing one PA are indicated by arrows, and the species containing two PA are indicated by dashed arrows.

Fig. 3.

MALDI-TOF MS/MS spectra of M. tuberculosis H37Rv Ac₂SGL are shown. A: Positive-ion mode MS/MS spectrum is shown of precursor ions [M+2Na]⁺ at m/z 1,295.9 of Ac₂SGL_1249.9. B: Positive-ion mode MS/MS spectrum is shown of precursor ions [M+2Na]⁺ at m/z 1,323.9 of Ac₂SGL_1277.9. C: Negative-ion mode MS/MS spectrum is shown of precursor ions [M-H]⁻ at m/z 1,277.9.

Fig. 4.

RP-HPLC purification of Ac₂SGL, monitored by off-line negative-ion mode MALDI-TOF-MS is shown. MALDI-MS mass spectra are shown of specific 1 ml fractions collected after injection of 50 µg of Ac₂SGL on C₁₈ Atlantis column. The fatty acid composition of Ac₂SGL species is annotated when deduced from MS/MS experiments. Stars indicate the matrix ion.

Fig. 5.

SGLs of a clinical M. tuberculosis CAS isolate. MALDI-TOF-MS spectra in negative-ion mode of the “Acetone-Soluble” fraction (A), and of purified Ac₄SGL (B), Ac₃SGL (C), Ac₂SGL (D) and Ac₁SGL (E).

Fig. 6.

Comparative MALDI-MS analyses of Ac₂SGL purified from both ΔmmpL8 mutants of M. tuberculosis and M. tuberculosis H37Rv are shown. Negative-ion mode MALDI-TOF-MS spectra of Ac₂SGL of mmpL8::hyg M. tuberculosis H37Rv Pasteur strain (A) and ΔmmpL8 jcm108 M. tuberculosis Erdman strain (B) and M. tuberculosis H37Rv (C) are shown. Expanded area (m/z 1,272–1.,284) is centered on the m/z 1,277.9 ion (D–F).

Fig. 7.

Schematic representation of the pathway of completion of SGL biosynthesis in M. tuberculosis H37Rv (WT) and ΔmmpL8 mutants of M. tuberculosis (ΔmmpL8) is shown [adapted from Bertozzi and Schelle (31) with permission]. The SGLs are not inserted in membrane, as they should be, for simplification and clarity purposes. Our results favor an extracellular acylation of Ac₂SGL, following the transport of Ac₂SGL by MmpL8. Indeed, the MmpL8 knockout strains also synthesize very weak amounts of Ac₃- and Ac₄SGL (mAc₃- and mAc₄SGL) but from a different structure. This model implies that HPA is transported by an unknown protein (annotated as “?”) to generate Ac₃- and Ac₄SGL species. Dashed insets show the novel SGL species described for the first time in this study. T₂S, trehalose-2′-sulfate.