PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor sulfolipid-1

Abstract

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.

Fig. 1.

SL-1, SL₁₂₇₈, and genomic organization. (A) Structure of SL-1. (B) Structure of SL₁₂₇₈. (C) The genomic arrangement of the SL-1 biosynthetic cluster. papA1 resides downstream of pks2, whereas papA2 lies further downstream of mmpL8.

Fig. 2.

PapA2 and PapA1 are acyltransferases that act sequentially on T2S. (A) PapA2 was incubated with radiolabeled PCoA and either trehalose (Tre) or T2S, and the reaction was analyzed by TLC. A product was observed only in the reaction with T2S (arrowhead). (B) FT-ICR MS of the PapA2 reaction. Synthetic trehalose-2-sulfate-2′-palmitate (SL₆₅₉) exhibits an m/z = 659.30. The same ion was observed in the PapA2 reaction (PapA2 rxn). The control reaction lacking PapA2 showed no product at m/z = 659. (C) PapA1 was incubated with radiolabeled PCoA and the product of the PapA2 reaction (PapA2 rxn). The reaction was analyzed by TLC, and a new product was observed (arrowhead). (D) FT-ICR MS of the PapA1 reaction. An ion with m/z = 897.54 was observed in the PapA1 reaction (PapA1 rxn), whereas the control reaction lacking PapA1 showed no such product.

Fig. 3.

papA2 and papA1 are essential for SL-1 biosynthesis in vivo. (A) WT M. tb, ΔpapA2, and ΔpapA1 were labeled with Na₂³⁵SO₄. The total lipids were extracted and analyzed by TLC. All three extracts show T2S. The ΔpapA2 extract lacks a spot corresponding to SL₆₅₉, the product of the PapA2 reaction in vitro. Both ΔpapA2 and ΔpapA1 extracts lack spots corresponding to SL₁₂₇₈ and SL-1. (B) FT-ICR MS analysis of total lipids from WT, ΔpapA2, and ΔpapA1. WT M. tb extracts display the characteristic SL-1 lipoforms, which are absent from the ΔpapA2 and ΔpapA1 extracts. (C) Identification of a peak corresponding to the exact mass of SL₆₅₉ in WT M. tb and ΔpapA1 samples. This metabolite is absent from ΔpapA2 extracts.

Fig. 4.

Disruption of papA2 or papA1 does not alter the virulence of M. tb in mice. BALB/c mice were infected via aerosol with WT (filled squares), ΔpapA2 (open circles), or ΔpapA1 (open triangles) strains at an initial inoculum of ≈250 bacteria per lung. Growth in the lungs (A), spleen (B), and liver (C) was monitored by harvesting organs at 0, 10, 23, and 44 days after infection. Bacillary loads were determined by plating dilutions on solid media. Each data point represents the average of cfu from four to five mice, and error bars indicate the standard deviation from the means.

Fig. 5.

Proposed SL-1 biosynthetic pathway. (A) Trehalose is first sulfated by Stf0 to form T2S. PapA2 then acylates the 2′-position of T2S to form SL₆₅₉. A (hydroxy)phthioceranic acid is then synthesized by Pks2 and transferred directly by PapA1 onto SL₆₅₉ to generate SL₁₂₇₈. (B) The diacylated SL₁₂₇₈ is then transferred by MmpL8 to the exterior of the cell where the final acylations occur.