The Mycobacterium tuberculosis pks2 Gene Encodes the Synthase for the Hepta- and Octamethyl-branched Fatty Acids Required for Sulfolipid Synthesis

Abstract

Multidrug-resistant tuberculosis is a major global health emergency. Cell wall lipids of Mycobacterium tuberculosis can play crucial roles in the pathogenesis. The enzymes involved in their synthesis can be ideal new drug targets against tuberculosis, because many such lipids are unique to this pathogen. A variety of multiple methyl-branched fatty acids are among such unique lipids. We have identified seven genes highly homologous to the mas gene, which is known to be involved in the production of one class of such multiple methyl-branched fatty acids. One of these mas-like genes, pks2, was disrupted using a phage-mediated delivery of the disruption construct. Gene disruption by homologous recombination was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Thin-layer and radio gas-chromatographic analyses of lipids derived from [1-14 C]propionic acid and gas chromatography/mass spectrometry analysis of the fatty acids and hydroxy fatty acids showed that the pks2 mutant was incapable of producing hepta-and octamethyl phthioceranic acids and hydroxyphthioceranic acids that are the major acyl constituents of sulfolipids. Consequently, pks2 mutant does not produce sulfolipids. Sulfolipid deficiency in pks2 mutant was confirmed by twodimensional thin-layer chromatographic analysis of lipids derived from [1-14 C]propionic acid and 35 SO 4 Ϫ2 . With this sulfolipid-deficient mutant, it should be possible to test for the postulated important roles for sulfolipids in the pathogenesis of M. tuberculosis.

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

“…Mt papA1 and papA2 are linked to pks2, a gene believed to be required for synthesis of the methyl-branched PK found esterified to trehalose in sulfolipids (19,45). Mt papA3 is linked to pks 3͞4, which are proposed to be required for synthesis of two methyl-branched PK moieties of polyacyltrehaloses (46).…”

“…Current models for PDIM synthesis propose involvement of at least three pks gene systems: (i) ppsA-E, required for POL and PONE synthesis (12); (ii) mas, required for mycocerosic (MYC) acid synthesis (12), and (iii) pks15͞1, required for incorporation of the phenolic group in mycoside PDIM variants (18). Recent evidence suggests that additional pks genes are involved in PDIM synthesis (8,10,19). Despite progress made since early studies on PDIM synthesis described 40 years ago (20,21), the enzyme(s) catalyzing diesterification of POL and PONE with MYC acid remains unknown.…”

Synthetic Studies toward Mycobacterium tuberculosis Sulfolipid-I

“…[8][9][10][11][12] By contrast, significant progress has been made in the last 5 years in understanding the biosynthesis of SL-I. [13][14][15][16][17][18][19][20][21] One of its presumed precursors appears to have a hydroxyphthioceranoyl group at the 3′ position (rather than the 6′ position as shown in Figure 1), however, casting some doubt on the accuracy of Goren's structure. 19,20 To investigate the biological activity of SL-I and to resolve questions concerning its precise structure, we set out to synthesize SL-I and analogs thereof.…”

A sulfated metabolite produced bystf3negatively regulates the virulence ofMycobacteriumtuberculosis

“…SL-1 has generated much interest because of its elaborate structure and the observation that its abundance correlates with strain virulence (15)(16)(17)(18)(19)(20)(21)(22). Advances in M. tuberculosis genetics and genome sequence data facilitated several contemporary studies that addressed aspects of the biosynthesis and the function of SL-1 in vivo (23)(24)(25)(26). Although these studies have greatly increased our understanding of SL-1, its function in the life cycle of the bacterium remains unknown.…”