Abstract
Genetic construction of a mutant strain (designated MSMEG4245) of Mycobacterium smegmatis, defective in a broadly conserved gene for a putative glycosyltransferase of the glycosyltransferase-C superfamily, results in a phenotype marked by the virtual absence of the phosphatidylinositol-containing lipomannan and lipoarabinomannan, replaced instead by a novel truncated form of lipomannan. The normal spectrum of phosphatidylinositol mannosides, long presumed precursors of these lipoglycans, was retained. Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry of the mutated form of lipomannan shows a family of phosphatidylinositol-anchored lipomannans with from only 5 to 20 Manp residues as compared with lipomannan from the wild type strain consisting of 21-34 Manp residues but with few changes in the branching pattern. Thus, MSMEG4245 is apparently a key mannosyltransferase, required for the proper elongation of lipomannan to its normal state and subsequent synthesis of lipoarabinomannan. The corresponding ortholog in Mycobacterium tuberculosis H37Rv has been identified as Rv2174. This previously unrecognized feature of the biosynthesis of lipomannan/lipoarabinomannan allows a significant revision of structural and biosynthetic schemata and provides a molecular basis of selectivity in biosynthesis, as conferred by the MSMEG4245 gene.
Highlights
PIM4 is regarded as the crucial intermediate linking the early phosphatidylinositol mannosides (PIMs) with LM/LAM, an assumption reinforced by the isolation of a M. smegmatis mutant unable to synthesize PIM6 but accumulating PIM4 [16]
The predicted structural features as well as the localization of MSMEG4245, close to MSMEG4250, a GT-C ManT involved in LM/LAM biosynthesis [17], suggest that MSMEG4245 represents a GT involved in LM/LAM biosynthesis
Our early demonstration [5] that the known soluble arabinomannans and mannans of mycobacteria are lipoglycans based on phosphatidylinositol, and structurally related to the PIMs most extensively described by Brennan and Ballou [9] in the 1960s, has generated considerable work on the role of these lipoglycans in innate immunity and the interaction of
Summary
It has been proposed that PIM6 is a “dead end” product, not involved in the biosynthesis of LM/LAM, as it contains two (1 3 2) linked Manp residues, a combination absent from LM/LAM [15]. Preliminary examination of the phenol extracts of whole cells by SDS-PAGE revealed the accumulation of a new major product, intermediate in size between LM and PIMs, referred to as mutLM (Fig. 2A).
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