The pimB gene of Mycobacterium tuberculosis encodes a mannosyltransferase involved in lipoarabinomannan biosynthesis

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was exami ned utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferas e genes involved in LAM synthesis, Cell-free reactions utilizing cellular m embranes 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 ge nomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) e ncoding a target of ManN would impart a ManN-resistant phenotype under thes e conditions. This strategy led to the identification of pimB, whose deduce d amino acid sequence shows similarity to mannosyltransferases and other gl ycosyltransferases. Partially purified recombinant PimB protein from Escher ichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of tria cylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidy linositol monomannoside.