Exquisite specificity of Pks13 defines the essentiality of trehalose in mycobacteria
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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, MAs are transported across the cell envelope in a sugar-esterified form, generally believed to be trehalose monomycolates (TMMs). However, 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 final biosynthetic steps affording TMMs. We show that in addition to TMMs, new MAs are also not produced in the absence of trehalose. During trehalose depletion, mycolate precursors accumulate on Pks13, the polyketide synthase catalyzing the condensation of MAs, and ligation to the sugar head group, possibly blocking the enzyme and preventing the synthesis of new mature MAs. We establish that the native mycolate precursors can only be released by trehalose in vitro, and subsequently processed in a final reduction step. Furthermore, we demonstrate that only trehalose can re-activate MA biosynthesis in trehalose-depleted cells. Our work reveals the exquisite specificity of Pks13 for trehalose, providing a compelling reason for the its essentiality in mycobacteria, as well as new ideas towards the future development of anti-mycobacterial strategies.
Significance Statement
Tuberculosis and other diseases caused by mycobacteria pose a persistent threat to human health and well-being. One reason why mycobacterial infections are hard to treat is the presence of the outer membrane, which serves as a barrier that confers resistance against multiple antibiotics. The mycobacterial outer membrane comprises mycolic acids, whose biosynthesis at late stages remains to be clarified. In this study, we characterize the penultimate step in mycolic acid biosynthesis, and discover extremely high specificity for the usage of the disaccharide trehalose by the synthase enzyme. Our work answers key questions in outer membrane biogenesis, and provides a molecular basis for why trehalose is essential for mycobacterial growth, a novel insight that is exploitable for future anti-mycobacterial strategies.
