Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach

Abstract

The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell-cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SO 4 2-). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.

Fig 1.

Structure of SL-1 (A) and mycothiol (B). The SL-1 structure shown is as originally proposed by Goren and colleagues (10, 35).

Fig 2.

General method for the identification of sulfated metabolites in microbes. (A) The sulfate assimilation pathway begins with the conversion of inorganic sulfate (SO) to APS by ATP sulfurylase (CysN,D). APS lies at a branchpoint in the pathway in mycobacteria (17). APS kinase (CysC) phosphorylates APS to form 3′-phosphoadenosine 5′-phosphosulfate (PAPS), the sulfate donor for sulfotransferases. These enzymes transfer the sulfuryl group onto a variety of substrates in the cell, forming sulfated molecules (red). In the reductive branch of the pathway, APS is reduced by APS reductase (CysH), eventually leading to the production of r-sulfur-containing molecules (blue). These can be distinguished from sulfated molecules (red) by using a mutation (green “×”) that blocks the reductive branch of the pathway. (B) MS can be used with heavy sulfur isotope labeling and the mutant in A (ΔcysH) to identify and distinguish r-sulfur and sulfated molecules. Sulfur-containing molecules are identified by MS in wild-type cells by virtue of their incorporation of ³⁴S upon growth in ³⁴SO–containing media (Step 1). Sulfated (red) molecules can be distinguished from r-sulfur-containing molecules (blue) by performing the same experiment using a ΔcysH strain (Step 2).

Fig 3.

Incorporation of ³⁴S into SL-1 can be detected in crude M. tuberculosis extracts. (A) Typical mass spectrum of M. tuberculosis extracts. The SL-1 lipoform envelope (bracketed) is readily observed in these samples. (B) Mass spectra of a purified SL-1 standard and a crude M. tuberculosis extract. (C) Zoom-in of one SL-1 lipoform showing that the mass of the molecule shifts by 2.00 Da in WT M. tuberculosis grown in ³²SO (Top) vs. ³⁴SO (Middle). This mass shift is also observed in M. tuberculosis ΔcysH grown in ³⁴SO + ³²met (Bottom), confirming that the compound is sulfated.

Fig 4.

Identification of previously unknown sulfated compounds in M. tuberculosis and M. smegmatis. (A) Mass spectra of M. smegmatis extracts showing that m/z 583.13 contains r-sulfur, whereas m/z 639.19 is sulfated. The m/z 583.13 ion (Left) shifts to m/z 585.12 in WT (Step 1) cells grown in ³⁴SO. The ion does not incorporate ³⁴S when ΔcysH is grown in ³⁴SO and ³²met (Step 2). The m/z 639.19 ion (Right) shifts to m/z 641.18 when WT (Step 1) and ΔcysH strains (Step 2) are grown in ³⁴SO. (B) Mass spectra of M. tuberculosis extracts showing m/z 881.57 is a sulfated compound. The m/z 881.57 ion shifts to m/z 883.57 when WT (Step 1) and ΔcysH strains (Step 2) are grown in ³⁴SO.

Fig 5.

MS/MS analysis of m/z 421.07 and a synthesized trehalose-2-sulfate standard. (A) Product ions of the m/z 421.07 ion. (B) Product ions of the trehalose-2-sulfate standard. (Inset) The assignment of product ions from A and B based on the structure of trehalose-2-sulfate.