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.
Highlights
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
We identified di-acylated sulfolipids, renamed SGLs (e.g., Ac2SGL [in AcXSGL, X refers to the total number of acyl groups, whatever the nature of the fatty acids, which could be either palmitic, stearic, hydroxyphthioceranoic, phthioceranoic, or oxophthioceranoic acids]), as new lipidic antigens presented by CD1b to T cells [14]
Goren et al previously characterized five SGL families, four of which were tetra-acylated (SL-I, SL-I′, SL-II, and SL-II′), and one was tri-acylated (SL-III) [3,4,5,6]. Those authors used an elegant strategy based on chemical degradation/modification and subsequent MS and infrared spectroscopy analyses that allowed i) identification of trehalose-2-sulfate, ii) determination of the acylated positions on the trehalose core, iii) identification of the acyl substituents (C16/C18, HPA, and PA), and iv) the determination that the 2-position of SL-I is acylated by one C16 or C18 and that the 3-position is occupied almost exclusively by Mycobacterium tuberculosis sulfolipids 1107 Q2 one PA [6]
Summary
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. 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. A complete family of mono-acylated SGLs was characterized in high abundance for the first time. Sulfoglycolipids (SGLs) were originally discovered in 1959 by Middlebrook et al [1] in human (H37Rv) and bovine (Vallée) virulent strains of Mycobacterium tuberculosis. Almost 2 decades later, Goren et al [2] showed that this family of lipids was specific to M. tuberculosis and proposed that their amount correlated with the virulence of the M. tuberculosis strain in the guinea pig model of infection.
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