Metabolic Network for the Biosynthesis of Intra- and Extracellular α-Glucans Required for Virulence of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis synthesizes intra- and extracellular α-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the α-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of α-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in α-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that α-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block α-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some α-glucan is exported to form the α-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate α-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADP-glucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying α-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of α-glucan could be constructed, showing a direct link between the GlgE pathway, α-glucan biosynthesis and virulence in a mouse infection model.

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice

“…These included the genes treX , treY and treZ , as expected, since they mediate de novo synthesis of trehalose from α-glucans with their combined inactivation with otsA resulting in trehalose auxotrophy [12]. ADP-glucose pyrophosphorylase GlgC, which is involved in α-glucan production [26], was also found to be essential only in the absence of trehalose suggesting that inactivation of glgC in the Δ otsA (u) mutant results in glucan deficiency and depletes the TreX-TreY-TreZ pathway of its substrate. The nature of these genetic interactions implies that the two other identified genes, Rv0907 and Rv3160c of unknown function, might also play a direct or indirect role in α-glucan biosynthesis.…”

“…We have very recently described the synthetic lethal interaction between otsA and glgA in M . tuberculosis , likely caused by toxic accumulation of the common precursor ADP-glucose [26]. Furthermore, trehalose recycling via the ABC transporter LpqY-SugABC becomes essential when the OtsA-OtsB2 pathway is blocked, likely by further depleting intracellular trehalose levels via secretion of trehalose during cell wall assembly.…”

“…This is not unusual as we have recently described a similar in vitro - in vivo difference in flux through alternative sugar metabolic pathways for biosynthesis of maltose-1-phosphate in M . tuberculosis [26]. …”

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae

“…It is therefore possible that the accumulation of glucose 1-phosphate and galactose 1-phosphate slows growth in S. venezuelae. Interestingly, the accumulation of ADP-glucose appears to be lethal in M. tuberculosis (37), so perhaps it is the accumulation of GDP-glucose that actually slows the growth of S. venezuelae . Either way, it is intriguing that the phenotype is cell density-dependent.…”

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…Furthermore, α-glucan modulates immune responses as a ligand of DC-SIGN [149] and alters the differentiation of monocyte-derived DCs and blocks CD1-expression, thereby inhibiting presentation of antigenic lipids of Mtb to CD1-restricted T cells [349]. Furthermore, Mtb mutants deficient in the synthesis of capsular glucan were less virulent and therefore unable to persist in vivo in mice [350,351]. In another mouse study, the capsular component arabinomannan (AM) could be detected in mouse tissue and its amount correlated with colony-forming units (CFU).…”