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 ( 34 SO 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.T he modification of primary and secondary metabolites by the addition or removal of sulfate can have a profound influence on their biological properties (1-5). Typically, sulfated molecules are directed outside the cell, where they serve as modulators of cell-cell interactions. As a notable example pertinent to human health, sulfation of the glycans of endothelial CD34 engenders high-affinity binding with the leukocyte adhesion molecule L-selectin, facilitating an interaction that eventually leads to the recruitment of lymphocytes into peripheral lymph nodes (6). Similarly, sulfation of tyrosyl residues found on the chemokine receptor CCR5 is a modification required for binding of HIV gp120 and therefore efficient viral entry (7).The roles of sulfated compounds in prokaryotes and other microbes are less clear. In one well-characterized case, however, sulfation acts as a modulator of cell-cell communication, similar to its role in eukaryotes. The nitrogen-fixing bacterium Sinorhizobium meliloti utilizes a sulfated glycolipid as a secondary messenger to induce root nodulation in its plant host alfalfa (4,8). Mutants lacking the sulfotransferase that installs this sulfate ester are unable to induce root nodulation in alfalfa but gain the ability to colonize the roots of vetch. That sulfation of a single glycolipid plays such a vital role in nitrogen fixation has far-reaching implications for the agricultural community and presents a possible target for chemical or genetic engineering.Sulfation may also be relevant to the process of bacterial pathogenesis (9). Several mycobacteria, including the human pathogens Mycobacterium tuberculosis and Mycobacterium avium, are known to produce sulfated compounds. One example is Sulfatide-1 (SL-1, Fig. 1A), a sulfated glycolipid from M....

A sulfated metabolite produced bystf3negatively regulates the virulence ofMycobacteriumtuberculosis

“…3A; ref. 29). When extracts of ⌬cysC were analyzed in this manner, they were found to specifically lack S881 (Fig.…”

“…S881 was initially identified in a mass spectrometric screen of M. tuberculosis extracts (29). The low abundance of S881 precluded detailed structural investigation; however, the presence of a sulfate ester was suggested by isotopic labeling and fragmentation analysis.…”

“…To test for potential attenuation of the WT strain, two additional mutants were generated and analyzed in parallel experiments by using identical methods. One of these mutants, a deletion of a nonessential gene, behaved as WT, and the other, a sulfur auxotroph (⌬cysH) (29,36), was attenuated relative to WT (Fig. 4A).…”

“…Although these studies have greatly increased our understanding of SL-1, its function in the life cycle of the bacterium remains unknown. Other sulfated molecules identified in mycobacteria include sulfated glycopeptidolipids present in the cell envelopes of M. avium (27) and M. fortuitum (28), and trehalose-2-sulfate (29) in M. smegmatis. The functions of these metabolites are also unknown.…”

“…The abundance of stf genes suggested that M. tuberculosis has the capacity to synthesize sulfated metabolites in addition to SL-1. Subsequently, mass spectrometric analysis of M. tuberculosis extracts revealed a previously undescribed sulfated compound (m͞z 881.56, now termed S881) that is structurally unrelated to SL-1 (29).…”