Biochemical Function of msl5 ( pks8 plus pks17 ) in Mycobacterium tuberculosis H37Rv: Biosynthesis of Monomethyl Branched Unsaturated Fatty Acids

Abstract

We show that the disruption of one of the mycocerosic acid synthase (mas)-like genes, msl5 (pks8 plus pks17) in Mycobacterium tuberculosis H37Rv generates a mutant incapable of producing monomethyl branched unsaturated C 16 to C 20 fatty acids that are minor constituents of acyltrehaloses and sulfolipids. The msl5 mutation did not cause any significant change in the acyl lipid composition and also did not affect growth in culture, in mouse alveolar macrophage cell line MH-S, or in the murine lung.Mycobacterium tuberculosis infection is present in a latent form in one-third of the world population, and 5 to 10% of them will probably develop active tuberculosis some time during their life. This disease accounts for more than a quarter of all preventable adult deaths (32). The high degree of success of this pathogen is, to a large extent, due to its ability to evade the natural host defenses and antimycobacterial therapy. The unusually lipid-rich (50 to 60%) cell wall of the pathogen, which constitutes an impermeable barrier, plays a major role in its ability to successfully infect its host (12,19,21,23). Reflecting the unusual variety and content of lipids in this organism, its genome contains an unusually large number of genes that show homology to genes involved in lipid metabolism (7). Many of these belong to the polyketide synthase (pks) family. These genes encode large multifunctional proteins that contain all of the domains required to catalyze the various steps involved in fatty acid synthesis. One of these, mycocerosic acid synthase, which catalyzes the synthesis of multiple methyl branched fatty acids, has been purified and characterized (22), and the gene that encodes this protein (mas) has been cloned (20). Because isolation and characterization of the many large proteins of similar size and similar functions would pose a technical challenge, a genetic approach has been used to identify the biochemical functions of the pks genes. Based on the lipids missing in the mutants in which specific pks genes have been disrupted, the biochemical functions of some pks genes have been deduced (3,6,9,14,24,(27)(28)(29). However, the biochemical functions of most pks genes remain unknown.Based on homology to catalytic domains involved in fatty acid synthesis, pks8 would encode ketoacyl synthase (KS), acyl transferase (AT), dehydratase (DH), and enoyl reductase (ER) domains, whereas the adjoining pks17 gene would encode a ketoreductase (KR) and acyl carrier protein (ACP) domain. Thus, the products of pks8 and pks17 together would contain a complete set of domains required to make a saturated fatty acid. Because of its similarity to mas, we designated this combination of pks8 and pks17 as msl5 (27). To elucidate the nature of the branched fatty acids generated by the msl5 gene product, we disrupted this gene and used [1-14 C]propionic acid as a radiotracer to identify the fatty acids missing in the msl5 mutant. We report that this approach identified the msl5 product as the one responsible for the synthesis of 2-methyl...

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

“…The Mt H37Rv genome contains 24 PK synthase genes (pks genes) organized in several loci (5,6), some of which are relevant for virulence (2,3,(7)(8)(9)(10)(11)(12)(13). Various genes associated with these loci encode proteins of unknown functions (5,6).…”

A Point Mutation in the Two-Component Regulator PhoP-PhoR Accounts for the Absence of Polyketide-Derived Acyltrehaloses but Not That of Phthiocerol Dimycocerosates in Mycobacterium tuberculosis H37Ra

“…as the solvent. The minor compound(s) displaying similar migration properties as DAT in the H37Ra strain might correspond to minor forms of diacyltrehaloses esterified with hydroxylated long-chain methyl-branched fatty acids or short-chain unsaturated mono-methyl-branched fatty acids (2,13). PDIM were analyzed with petroleum ether (60/80°C):ethyl acetate (98:2, by vol., three developments) as the solvent.…”

“…Radiolabeling of whole M. tuberculosis cells with [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]propionate (specific activity, 56.7 Ci mol Ϫ1 , MP Biomedicals Inc.), extraction and analyses of lipids were performed as described (16). Neutral red staining of tubercle bacilli was performed as described by Soto and collaborators (36).…”

Induction of a Novel Class of Diacylglycerol Acyltransferases and Triacylglycerol Accumulation in Mycobacterium tuberculosis as It Goes into a Dormancy-Like State in Culture

“…Fatty acid composition of labeled TG was determined by radio-gas chromatography (radio-GC) of the total methyl esters or after argentation TLC (11). TGS activity in cell extracts from M. tuberculosis subjected to hypoxia and NO treatment.…”