THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria

Jonathan A.G. Cox, Katherine A. Abrahams, Carlos Alemparte, Sonja Ghidelli-Disse, Joaquín Rullas, Iñigo Angulo-Barturen, Albel Singh, Sudagar S. Gurcha, Vijayashankar Nataraj, Stephen Bethell, Modesto J. Remuiñán, Lourdes Encinas, Peter J. Jervis, Nicholas C. Cammack, Apoorva Bhatt, Ulrich Kruse, Marcus Bantscheff, Klaus Fütterer, David Barros, Lluis BallellGerard Drewes, Gurdyal S. Besra

Research output: Contribution to journalArticle

Abstract

Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.

Original languageEnglish
Article number15006
JournalNature Microbiology
Volume1
DOIs
Publication statusPublished - 18 Jan 2016

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    Cox, J. A. G., Abrahams, K. A., Alemparte, C., Ghidelli-Disse, S., Rullas, J., Angulo-Barturen, I., Singh, A., Gurcha, S. S., Nataraj, V., Bethell, S., Remuiñán, M. J., Encinas, L., Jervis, P. J., Cammack, N. C., Bhatt, A., Kruse, U., Bantscheff, M., Fütterer, K., Barros, D., ... Besra, G. S. (2016). THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria. Nature Microbiology, 1, [15006]. https://doi.org/10.1038/nmicrobiol.2015.6