Poce, G. | bioRxiv Science

bioRxiv2024

Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA

The peptidoglycan pathway represents one of the most successful antibacterial targets with the last critical step being the flipping of carrier lipid, undecaprenyl phosphate (C55-P), across the membrane to re-enter the pathway. This translocation of C55-P is facilitated by DedA and DUF368 domain-containing family membrane proteins via unknown mechanisms. Here we employ native mass spectrometry to investigate the interactions of UptA, a member of the DedA family of membrane protein from Bacillus subtilis, with C55-P, membrane phospholipids and cell wall-targeting antibiotics. Our results show that UptA, expressed and purified in E. coli, forms monomer-dimer equilibria, and binds to C55-P in a pH-dependent fashion. Specifically, we show that UptA interacts more favourably with C55-P over shorter-chain analogues and membrane phospholipids. Moreover, we demonstrate that lipopeptide antibiotics, amphomycin and aspartocin D, can directly inhibit UptA function by out-competing the substrate for the protein binding, in addition to their propensity to form complex with free C55-P. Overall, this study shows that UptA-mediated translocation of C55-P is potentially mediated by pH and anionic phospholipids and provides insights for future development of antibiotics targeting carrier lipid recycling.

molecular biology↗

bioRxiv2017

MmpL3 is the flippase for mycolic acids in mycobacteria

The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which in part render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. While the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. MmpL3, an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several anti-mycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria, and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism-of-action of BM212 as a potent MmpL3 inhibitor, and employ it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future anti-tuberculosis drugs.

biochemistry↗