In Vivo Biosynthesis of Terpene Nucleosides Provides Unique Chemical Markers of Mycobacterium tuberculosis Infection

Abstract

Summary Although small molecules shed from pathogens are widely used to diagnose infection, such tests have not been widely implemented for tuberculosis. Here we show that the recently identified compound, 1-tuberculosinyladenosine (1-TbAd), accumulates to comprise > 1 percent of all M. tuberculosis lipids. In vitro and in vivo, two isomers of TbAd were detected that might serve as infection markers. Using mass spectrometry and NMR, we established the structure of the previously unknown molecule, N6-tuberculosinyladenosine (N6-TbAd). Its biosynthesis involves enzymatic production of 1-TbAd by Rv3378c followed by conversion to N6-TbAd via the Dimroth rearrangement. Intact biosynthetic genes are observed only within M. tuberculosis complex bacteria, and TbAd was not detected among other medically important pathogens, environmental bacteria and vaccine strains. With no substantially similar known molecules in nature, the discovery and in vivo detection of two abundant terpene nucleosides support their development as specific diagnostic markers of tuberculosis.

Heterologous production of 1-tuberculosinyladenosine inMycobacterium kansasiimodels pathoevolution towards the transcellular lifestyle ofMycobacterium tuberculosis

“…Whereas the M. kansasii ::EV control did not release compounds that comigrated with either 1-TbAd or N 6 -TbAd, M. kansasii:Rv3377-78c produced high intensity (6.7-7.0 x10 6 counts) signals ( m/z 540.354) matching the expected retention time and mass ( m/z 540.3544) of the proton adducts of 1-TbAd and N 6 -TbAd. The extractions were performed at a range of pH (4.5 – 7.4) since both the Dimroth reaction that generates N 6 -TbAd and the capture of lysosomotropic agents are sensitive to pH, as previously explained (18, 22, 23). However, there was no clear impact of altering pre-extraction pH for two hours to the release nor relative abundance of 1-TbAd and N 6 -TbAd, suggesting that such effects, if existent, did not occur under the tested conditions (Fig.…”

“…Together, the two enzymes are responsible for the conversion of geranylgeranyl pyrophosphate (GGPP) into the recently identified M. tuberculosis -specific lipid 1-tuberculosinyladenosine (1-TbAd), which is a potential diagnostic molecular marker for TB disease (17–20). 1-TbAd further undergoes a chemical rearrangement, known as the Dimroth reaction, to generate N 6 -TbAd (18). While GGPP is found in both species and used as an intermediate in the biosynthesis of 1-TbAd by M. tuberculosis (17, 19), it is part of the biosynthetic pathway for the production of carotenoid pigments of M. kansasii , giving its characteristic yellow colour (21).…”

Antiinfectives targeting enzymes and the proton motive force

“…5A) and related compounds (75). Transposon mutants of Rv3378c show inhibited phagosomal acidification, and Rv3378c is necessary for production of TbAd and related metabolites.…”

Stereoselective Synthesis of 1-Tuberculosinyl Adenosine; a Virulence Factor of Mycobacterium tuberculosis

“…This result concluded our total synthesis of the naturally occurring tuberculosinyl adenosines and directly confirmed the existence of a plausible, nonenzymatic basis for generation of the N 6 -product from 1-TbAd, as hypothesized previously. 7 …”

“…6 Additional studies identified the pseudoisomer N 6 -tuberculosinyl adenosine ( N 6 -TbAd, Figure 1), which was postulated to arise from an in vivo Dimroth rearrangement of 1-TbAd. 7 …”