Abstract
Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.
Highlights
Contrast, relatively little is known about the biosynthesis of other prominent M. tuberculosis glycolipids, such as di, tri, and polyacyltrehaloses
These acyltrehaloses are located in the outer surface of the cell wall and contain di- and tri-methyl branched fatty acids that are only found in pathogenic species of mycobacteria [6, 7]
Genomic Analysis of the PAT Biosynthetic Locus—The pap gene family encodes polyketide synthase-associated acyltransferases that are involved in the synthesis of some of the complex lipids produced by M. tuberculosis [5, 11]
Summary
Reagents and Chemicals—Pfu DNA polymerase was from Stratagene (La Jolla, CA). Oligonucleotides were from Elim Biopharmaceuticals, Inc. (Hayward, CA). DNA sequencing was performed by Elim Biopharmaceuticals, Inc. 14C-Palmitoyl coenzyme A (14C-PCoA), 14Cpalmitic acid, 14C-butyryl coenzyme A, 14C-crotonoyl coenzyme A, and 14C-docosanoyl coenzyme A were purchased from ARC Radiolabeled Chemicals (St. Louis, MO; 50 –55 mCi/ mmol). The protein was purified using a gradient of 1.5–100% high salt buffer (50 mM Tris, pH 7.4, 1 M NaCl, 1 mM DTT, 1 mM TCEP, 10% glycerol) over 40 min at a constant temperature of 4 °C. 12746 JOURNAL OF BIOLOGICAL CHEMISTRY vector pMV261 [16] under the control of the groEL promoter, resulting in the complementation plasmid pMWS149 This plasmid was electroporated into the ⌬papA3 strain, and transformants were selected on kanamycin-containing plates. The remaining cell pellets were extracted with 4 ml of chloroform: methanol (1:1)
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