Abstract
The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.
Highlights
The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis
Little is known about the biosynthesis of LAM, structural similarities have suggested that the early precursors are phosphatidyl-myo-inositol (PI) and certain phosphatidylinositol mannosides (PIMs) [12,13,14], and this hypothesis is supported by recent direct biosynthetic evidence [15, 16]
Effect of Mannosamine on in Vitro PIM Synthesis—ManN (2-deoxy-2-amino-D-mannose) inhibits the synthesis of glycosylphosphatidylinositol anchors in Trypanosoma by chain termination, forming ManN-Man-GlcN-PI that cannot be further mannosylated at the 2-position of ManN [36]. This mode of action suggested that ManN might be a useful tool to study PIM biosynthesis in mycobacteria, and so it was applied to the cell-free system of Besra et al [15] that permits mannolipid synthesis in the presence of enzymatically active membranes from M. smegmatis
Summary
The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. This mode of action suggested that ManN might be a useful tool to study PIM biosynthesis in mycobacteria, and so it was applied to the cell-free system of Besra et al [15] that permits mannolipid synthesis in the presence of enzymatically active membranes from M. smegmatis.
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