Mycobacterium tuberculosis produces numerous exotic
lipids that have been implicated as virulence determinants. One such
glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four
lipid moieties. A diacylated intermediate in
SL-1 biosynthesis, SL(1278), has been shown to activate the adaptive immune response in human patients. Although several
proteins involved in
SL-1 biosynthesis have been identified, the
enzymes that acylate the T2S core to form SL(1278) and
SL-1, and the biosynthetic order of these acylation reactions, are unknown. Here we demonstrate that
PapA2 and PapA1 are responsible for the sequential acylation of T2S to form SL(1278) and are essential for
SL-1 biosynthesis. In vitro, recombinant
PapA2 converts T2S to 2'-palmitoyl T2S, and PapA1 further elaborates this newly identified
SL-1 intermediate to an analog of SL(1278). Disruption of
papA2 and papA1 in M.
tuberculosis confirmed their essential role in
SL-1 biosynthesis and their order of action. Finally, the Delta
papA2 and Delta papA1 mutants were screened for virulence defects in a mouse model of
infection. The loss of
SL-1 (and SL(1278)) did not appear to affect bacterial replication or trafficking, suggesting that the functions of
SL-1 are specific to human
infection.