Abstract
The biosynthesis of mycobacterial mannose-containing lipoglycans, such as lipomannan (LM) and the immunomodulator lipoarabinomanan (LAM), is carried out by the GT-C superfamily of glycosyltransferases that require polyprenylphosphate-based mannose (PPM) as a sugar donor. The essentiality of lipoglycan synthesis for growth makes the glycosyltransferase that synthesizes PPM, a potential drug target in Mycobacterium tuberculosis, the causative agent of tuberculosis. In M. tuberculosis, PPM has been shown to be synthesized by Ppm1 in enzymatic assays. However, genetic evidence for its essentiality and in vivo role in LM/LAM and PPM biosynthesis is lacking. In this study, we demonstrate that MSMEG3859, a Mycobacterium smegmatis gene encoding the homologue of the catalytic domain of M. tuberculosis Ppm1, is essential for survival. Depletion of MSMEG3859 in a conditional mutant of M. smegmatis resulted in the loss of higher order phosphatidyl-myo-inositol mannosides (PIMs) and lipomannan. We were also able to demonstrate that two other M. tuberculosis genes encoding glycosyltransferases that either had been shown to possess PPM synthase activity (Rv3779), or were involved in synthesizing similar polyprenol-linked donors (ppgS), were unable to compensate for the loss of MSMEG3859 in the conditional mutant.
Highlights
Tuberculosis (TB) affects a third of mankind and causes 1.7 million fatalities annually [1]
In an effort to first confirm the in vivo role of Mt-Ppm1/D2 in LM/LAM biosynthesis we aimed to test the essentiality of polyprenylphosphate-based mannose (PPM) glycosyltransferase activity in M. smegmatis by using CESTET, a genetic tool for testing gene essentiality in M. smegmatis
As MSMEG3859 was shown to be sufficient for the enzymatic generation of PPM in vitro, we reasoned that the PPM synthase-encoding MSMEG3859, but not the membrane segment-encoding MSMEG3860, was an essential gene
Summary
Tuberculosis (TB) affects a third of mankind and causes 1.7 million fatalities annually [1]. A particular group of specialized glycophospholipids, phosphatidylmyo-inositol (PI) mannosides (PIMs) and lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), are found in the outer leaflet of mAGP [4]. LM and LAM, which are based on a core PIM unit, possess an elongated a(1R6) linear, a(1R2) branched mannan, of approximately 30 mannopyranose (Manp) residues, and linked to its terminus to a branched D-arabinan domain of approximately 70 arabinofuranose (Araf) residues, assembled through several distinct structural motifs [4,5,6]. The large arabinan domain is capped to various degrees with short a(1R2) Manp chains in the case of M. tuberculosis [7], whereas in M. smegmatis, caps of inositol phosphate are present and termed PILAM [4], and M. chelonae possess non-capped LAM [8].
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