Cardiolipin modulation of MmpL3 in mycobacteria

Abstract

The complex cell envelope of mycobacteria is characterized by the presence of a unique outer membrane (OM) rich in mycolic acids (MAs). These long-chain, branched fatty acids confer extreme hydrophobicity to the OM, in part rendering the mycobacterial envelope impermeable to antibiotics and host defences. How MAs are transported from the inner membrane (IM) to the OM is largely unknown. The integral membrane protein MmpL3 plays an essential role in this process, but the mechanism(s) by which it exploits the proton motive force to flip and/or release MAs at the IM, in the form of trehalose monomycolates (TMMs), remains elusive. Here, we reconstitute and quantify the proton translocation activity of MmpL3 in artificial lipid bilayers, and discover a novel role for the phospholipid species, cardiolipin (CL), in regulating MmpL3 function. We find that mutations in conserved residues, or binding of known inhibitors in the central channel of MmpL3 do not diminish proton translocation activity. Instead, the specific presence of CL abolishes proton translocation by MmpL3 in vitro. Furthermore, we establish that an MmpL3 variant containing substitutions in a CL-binding site predicted in silico is no longer modulated by CL in vitro, and is unable to support growth of Mycobacterium smegmatis. Our work provides previously unappreciated insights into lipid regulation of MmpL3 activity in mycobacteria, and expands the guiding principles for the development of anti-mycobacterial inhibitors targeting this essential transporter. SignificanceMycobacterial species, including the human pathogen Mycobacterium tuberculosis, are surrounded by a double-membrane cell envelope that makes them intrinsically resistant to many antibiotics. Specifically, the outer membrane (OM) contains unique lipids called mycolic acids (MAs), whose transport pathway across the envelope is poorly understood. In this study, we characterized the biochemical activity of the essential MA transporter MmpL3, and uncovered a novel mechanism for lipid-mediated functional regulation. Our work highlights the importance of protein-lipid interactions in defining transporter activity, provides insights into MA transport and OM assembly in mycobacteria, and sets the stage for the development of anti-mycobacterial strategies.