Exquisite specificity of Pks13 defines the essentiality of trehalose in mycobacteria

Abstract

Mycolic acids (MAs) are the major component of the mycobacterial outer membrane, a key contributor to the intrinsic resistance of mycobacteria to external insults including multiple antibiotics. After being synthesized in the cytoplasm and before reaching the outer membrane, MAs are transported across the inner membrane in the form of an acylated sugar, generally believed to be trehalose monomycolates (TMMs). Whether trehalose is the only mycolate carrier during transport is under debate, and why this highly abundant disaccharide is essential for mycobacterial growth is unclear. To address these questions, we leveraged a trehalose auxotrophic Mycobacterium smegmatis strain to investigate the biosynthetic steps affording TMMs. We show that in addition to TMMs, MAs are also not produced in the absence of trehalose. This is likely due to a product inhibition mechanism where un-reduced MA precursors accumulate on Pks13, the protein catalyzing the ligation of mycolic acids and the sugar head group. We establish that the un-reduced mycolates could only be released by trehalose, revealing exquisite Pks13 specificity, and subsequently reduced by CmrA in vitro. Furthermore, only trehalose and its analogs can reactivate MA biosynthesis in cells. Finally, by replacing trehalose with a 6-deoxy analog in cells, we demonstrate that the cord factor trehalose dimycolate is dispensable for M. smegmatis growth in vitro. Our work gives a clear depiction of how TMMs are formed and provides a compelling reason for the essentiality of trehalose, shedding light on the development of future anti-mycobacterial strategies. Significance StatementTuberculosis and other diseases caused by mycobacteria pose a persistent threat to human health and well-being. The mycobacterial outer membrane is a highly impermeable barrier, conferring intrinsic resistance to multiple antibiotics. How the biosynthesis of mycolic acids, the major component of the mycobacterial outer membrane, is driven by trehalose is unknown. In this report, we study the molecular basis for the essentiality of trehalose. We define the order of two critical steps in the mycolic acid biosynthetic pathway involving trehalose and demonstrate the highly specific role that trehalose plays therein. This work answers key questions in terms of outer membrane biogenesis in mycobacteria, providing a venue for the development of novel anti-mycobacterial strategies.