Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c

Significance Whereas most mycobacteria do not cause disease, Mycobacterium tuberculosis kills more than one million people each year. To better understand why Mycobacterium tuberculosis is virulent and to discover chemical markers of this pathogen, we compare its lipid profile with that of the attenuated but related mycobacterium, Mycobacterium bovis Bacillus Calmette–Guérin. This strategy identified a previously unknown Mycobacterium tuberculosis-specific lipid, 1-tuberculosinyladenosine, which is produced by the Rv3378c enzyme. The crystal structure of Rv3378c provides information supporting drug design to inhibit prenyl transfer. Discovery of 1-tuberculosinyladenosine provides insight into how Mycobacterium tuberculosis resists killing in macrophages and a new target for diagnosing tuberculosis disease. To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette–Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.