PapA1 and PapA2 are acyltransferases essential for the biosynthesis of theMycobacterium tuberculosisvirulence factor Sulfolipid-1

Abstract

pathogenesis ͉ biochemistry ͉ glycolipid ͉ sulfation T he thick Mycobacterium tuberculosis (M. tb) cell wall consists of numerous glycolipids that are distinctive to the mycobacterial genus, including phosphatidylinositol mannosides, trehalose mycolates, and lipoarabinomannans (1). These molecules are essential for many of the characteristics that distinguish mycobacterial pathogenesis, such as the inhibition of phagosomal maturation, drug resistance, and alteration of the host immune response (2-6). A family of cell surface sulfated lipids (dubbed sulfatides) were identified in M. tb extracts and correlated to strain virulence (7-9). The most abundant sulfatide, termed Sulfolipid-1 (SL-1), consists of a trehalose core, four fatty acyl groups, and a sulfate ester (Fig. 1A) (10-13). Despite the discovery of SL-1 nearly 50 years ago, the biological function of the molecule is not known. Conflicting reports suggest a role for SL-1 in superoxide (O 2 Ϫ ) release from human neutrophils or monocytes, alteration of trehalose dimycolate toxicity, and inhibition of trehalose dimycolate-induced macrophage recruitment (14-19). The relevance of these studies to the physiological role of SL-1 in M. tb infection is debatable.Although the role of SL-1 remains elusive, advances in genetics and metabolite analysis have sped the discovery of genes, proteins, and intermediates associated with SL-1 biosynthesis (20). Currently, three proteins are known to be involved in SL-1 assembly: Stf0, Pks2, and MmpL8. The sulfotransferase Stf0 sulfates trehalose at the 2-position, forming trehalose-2-sulfate (T2S), thereby initiating SL-1 biosynthesis (21). Meanwhile, the polyketide synthase Pks2 synthesizes the phthioceranoyl and hydroxyphthioceranoyl lipids that occupy the 6-, 6Ј-, and 3Ј-positions of SL-1 (Fig. 1 A) (22). The proteins responsible for transfer of the Pks2 products and the palmitoyl group to the T2S core, and the order in which these lipids are added, have not yet been defined.Insight into the order of lipid addition came from characterization of the putative lipid transporter MmpL8 (23,24). A mutant strain, ⌬mmpL8, lacks SL-1 but accumulates the diacylated intermediate SL 1278 (named for its observed mass) inside the cell (Fig. 1B). This intermediate possesses two of the four SL-1-associated lipids: a hydroxyphthioceranoyl group at the 3Ј-position and a palmitoyl group at the 2Ј-position (24). SL 1278 was recently found to be an immunostimulant in human tuberculosis patients (25). The glycolipid is presented on the surface of M. tb-infected antigen-presenting cells by CD1b, a member of the MHC class I-like CD1 family. Intriguingly, the ⌬mmpL8 mutant, which lacks SL-1 but accumulates SL 1278 , shows attenuated virulence in mice (23,24). By contrast, a ⌬pks2 mutant, which lacks both SL-1 and SL 1278 , is indistinguishable from WT M. tb in mice and guinea pigs (23,26). These observations suggest that SL 1278 , and possibly other SL-1 intermediates, modulate M. tb pathogenesis.In our effort to define the functions of M. tb sulf...

Deciphering sulfoglycolipids of Mycobacterium tuberculosis

“…The identifi cation of this mono-acylated species was unexpected, as the fi rst acylation step is considered to be a palmitoylation/stearoylation on the 2-position of the sulfated trehalose ( Fig. 1A ) and because PapA1, the acyl-transferase that adds the HPA on C2-palmitoyl/stearoyl-sulfated trehalose, has been shown to harbor no activity toward sulfated trehalose ( 25 ). More clues about the biosynthetic pathway of SGLs arose from an in-depth analysis of SGLs produced by D mmpL8 mutants.…”

“…Palmitate or stearate acyl chains derive from fatty-acid synthase-I (FAS-I), whereas HA and HPA are synthesized by polyketide synthase 2 (Pks2) ( 27 ) working in conjunction with the FadD23 fattyacyl-CoA ligase ( 28,29 ). Then, two acyl-transferases, PapA1 on one hand and PapA2 associated with Pks2 on the other hand, would act sequentially ( 25,30 ), where PapA2 converts sulfated trehalose into 2-palmitoyl/stearoyl-sulfated trehalose, and PapA1 further elaborates Ac 2 SGL by adding a methyl-branched fatty acid on the 3-position of trehalose. The MmpL8 protein, predicted to be a lipid transporter, is described as playing a role in mediating the transport of the Ac 2 SGL precursor forms from the cytosol into the cell envelope ( 13,15 ).…”

Bacterial immunostat: Mycobacterium tuberculosis lipids and their role in the host immune response

“…The polyketide synthase Pks2 synthesizes methyl-branched (hydroxy) phthioceranoyl 65 and PapA1 transfers the product of Pks2 to the 3-position of monoacylated-SGL, yielding Ac 2 SGL 71 . MmpL8 may flip Ac 2 SGL across the plane of the cell membrane, allowing it to be further modified to mature SL-1 before delivery to the cell wall 72 (Figure 1).…”

“…The acyltransferase PapA2 then catalyzes the esterification of T2S at the 2-position to generate a monoacylated intermediate monoacylated-SGL 71 . The polyketide synthase Pks2 synthesizes methyl-branched (hydroxy) phthioceranoyl 65 and PapA1 transfers the product of Pks2 to the 3-position of monoacylated-SGL, yielding Ac 2 SGL 71 .…”

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.…”