Mycobacterium tuberculosis releases an antacid that remodels phagosomes

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Abstract

Mycobacterium tuberculosis (Mtb) is the world’s most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.

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Fig. 1: Testing signaling-dependent and signaling-independent effects of TbAd on human cells.
Fig. 2: Quantitative measurements of 1-TbAd using an internal standard on a per cell basis.
Fig. 3: Effect of synthetic analogs of 1-TbAd on lysosomal pH in THP-1 cells and macrophages.
Fig. 4: 1-TbAd is a lysosomotropic antacid.
Fig. 5: Transmembrane permeation by TbAd analogs.
Fig. 6: Infection of human macrophages for 4 d with Mtb or the Rv3378c– deletion mutant, which lacks TbAd production.

Data availability

Institutional review boards require confidentiality of patient data and biological material. Distribution of Mtb strains is subject to biosafety approvals. Otherwise, all data and reagents are available.

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Acknowledgements

The authors thank H. van Veen and W. Tigchelaar for EM, P. Reinink for phylogenetic graphs and S. Suliman for advice. Work was supported by grant nos. AI116604 (to D.B.M. and N.N.v.d.W.), AI111224 (to D.B.M. and M.M.), GM065307 (to E.O.), CA158191 (to E.O.) and AI114952 (to S.T.), the Dutch Science Foundation NWO-VICI 70.57.443 (to A.J.M.) and a Canadian Institute of Health Research Foundation grant 148362 (to M.A.B.).

Author information

H.B., R.C. and M.M. carried out the patient studies. T.Y.C. carried out the confocal microscopy. T.Y.C. and T.E. carried out the flow cytometry. M.A.B., M.G., J.W., M.B.R. and D.C.Y. carried out the M. kansasii assays. S.R., A.K.R. and E.L. performed the lipid analysis. J.A.M. performed the statistical analysis. J.B., A.R.P., A.M. and B.B.S. performed the chemical synthesis. E.O. and X.F. did the IMV studies. N.N.v.d.W. and A.E.G. performed the EM. S.R. carried out the gene deletion. S.R., N.S. and S.W. carried out the infection studies. S.T.carried out the pH measurements. The experiments were designed by D.B.M., E.J.R., M.A.B., E.O., N.N.v.d.W., S.E., A.J.M., T.Y.C., S.T. and J.B. The manuscript was written by D.B.M., J.B. and A.J.M., with contributions from all authors.

Correspondence to D. Branch Moody.

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Synthetic procedures

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