A Chlamydia trachomatis UDP-N-acetylglucosamine acyltransferase selective for myristoyl-acyl carrier protein - Expression in Escherichia coli and formation of hybrid lipid A species

Chlamydia trachomatis lipid A is unusual in that it is acylated with myrist oyl chains at the glucosamine 3 and 3 ' positions, We have cloned and expre ssed the gene encoding UDP-N-acetylglucosamine 3-O-acyltransferase of C, tr achomatis (CtlpxA), the first enzyme of lipid A biosynthesis. C. trachomati s LpxA displays similar to 20-fold selectivity for myristoyl-ACP over R/S-3 -hydroxymyristoyl-ACP under standard assay conditions, consistent with the proposed structure of C, trachomatis lipid A. CtLpxA is the first reported UDP-N-acetylglucosamine acyltransferase that prefers a non-hydroxylated acy l-ACP to a hydroxyacyl-ACP. When CtlpxA was expressed in RO138, a temperatu re-sensitive lpxA mutant of Escherichia coli, five new hybrid lipid A speci es were made in vivo after 2 h at 42 degreesC, in place of Escherichia coli lipid A. These compounds were purified and analyzed by matrix-assisted las er desorption ionization/time of flight mass spectrometry. In each case, a myristoyl chain replaced one or both of the ester linked 3-hydroxymyristoyl residues of E. coil lipid A. With prolonged growth at 42 degreesC, all the eater-linked 3-hydroxymyristoyl residues were replaced with myristate chai ns. Re-engineering the structure of E. coil lipid A should facilitate the m icrobiological production of novel agonists or antagonists of the innate im munity receptor TLR-4, with possible uses as adjuvants or anti-inflammatory agents.