## 4 Mmpl3 Transporter: The Other Hot TB Target of the Moment  
### Mmpl3: A Mycolic Acid Transporter  
MmpL3 is a membrane protein, member of the resistance-modulation-cell division (RND) superfamily of transporters. In mycobacteria, MmpL transporters have specialized in the export of several lipids and glycolipids across the plasma membrane to the cell surface, namely, trehalose monomycolates (TMM), di- and poly-acyltrehaloses, sulfolipids, phthiocerol dimyococcates, monomycolyldiacylglycerol, glycolopeptideolipids and mycobacteria [74; 75; 76; 77; 78]. Genetic studies with transposon mutant libraries and the inability to knock-out the gene using different strategies suggested that MmpL3 was essential for the survival of _M. tuberculosis_[79; 38]. An alternative strategy, utilizing knock-down strains both in vitro and in vivo had confirmed that MmpL3 is indeed essential for survival. Silencing of MmpL3 in mice, both during the acute or persistence phase of infection, led to a complete clearance of bacteria from lungs and spleens. These studies not only reinforce the idea of MmpL3 as an attractive drug target but also the potential of MmpL3 inhibitors to shorten TB treatment [80]. MmpL3 contains 12 transmembrane segments with two periplasmic loops and one C-terminal cytoplasmic domain. The periplasmic loops are essential for the MmpL3 function and are involved in substrate binding. On the other hand, the cytoplasmic loop, which is only present in MmpL3 and MmpL11 but not in other members of the MmpL family, is not essential for MmpL3 transport activity [81]. The exact role of the C-terminal domain is still not clear but it has been proposed to act as a signal for polar localization of the protein, since its removal caused MmpL3 to be more diffuse across the cells in contrast to the typical subpolar localization of the full protein [82]. In addition to that, this domain was shown to be involved in protein-protein interactions with itself as well as with other proteins, pointing to a role in oligomerization and/or coordination of synthesis of other cell wall components [83]. MmpL3 transports TMM across the cell membrane to the periplasmic space where it serves as a mycolic acid donor to another TMM molecule to form TDM, or to arabinogalactan as part of the mAGP core. Both of these reactions are catalyzed by the Ag85 family of enzymes. Mycolic acids are essential components of the mycobacterial cell wall and its biosynthesis has been shown as an effective drug target in mycobacteria, with several antibiotics targeting this pathway. The best example of this is isoniazid, which inhibits the FASII enoyl-ACP reductase InhA, and is one of the pillars in tuberculosis treatment [84]. MmpL3 relies on the proton motive force as an energy source to drive TMM transport. Thus, the flux of protons across the transmembrane region of MmpL3 would drive the conformationalchanges necessary for the translocation of TMM. This is evidenced by mutagenesis studies showing that charged residues lying on the transmembrane region are essential for MmpL3 function and are most likely involved in this proton relay pathway. The mechanism and extent of TMM transport mediated by MmpL3 is still not fully understood. Spherolasts assays have suggested that MmpL3 acts as a flippase [15]. This conclusion was derived from the accumulation of TMM in the inner leaflet of the plasma membrane after MmpL3 inhibition. Although this would point to MmpL3 acting as a flippase, there is the possibility that MmpL3 inhibition would affect also other proteins acting upstream in the transport pathway and/or TMM biosynthetic enzymes. This holds true for other MmpL transporters where disruption of the transport leads to the accumulation of biosynthetic precursors and, in some cases, the complete abolition of the synthesis of the substrate [74; 78; 85; 86]. The recently solved crystal structure of MmpL3 has shed some light as to the possible mechanism of TMM translocation [25; 87]. Crystals of MmpL3 from _M. smegmatis_ were diffracted to a 2.59 A resolution.