MmpL7 Domain 2 Acts as a Dominant Negative Inhibitor of PDIM Synthesis In Vivo

<div><p>(A) The indicated strains carrying either no plasmid (−), control vector (vector), or a plasmid with MmpL7 domain 2 under the control of the constitutive <i>groEL2</i> promoter (MmpL7^d2) were labeled with ¹⁴C-propionate. Surface-exposed lipids (S) were extracted by resuspension in hexanes, and cell pellets (P) were harvested by centrifugation. Lipids from both fractions were extracted and separated by TLC under solvent conditions to separate PDIM (upper panel, keto and methoxy forms) and SL-1 (lower panel).</p><p>(B) Top: lipids were extracted as in (A) from pellets of wild-type cells carrying either no plasmid (−), the MmpL7 domain 2 expression plasmid (d2), or the MmpL7 domain 2 expression plasmid with the I611S mutation (d2-I611S). Bottom: Western blot analysis was performed to confirm equivalent expression of wild-type MmpL7 domain 2 and the I611S mutant by using antibodies against the hemagglutinin epitope tag.</p><p>(C) Complementation of an <i>mmpL7⁻ M. tuberculosis</i> strain with the wild-type (<i>mmpL7^wt</i>) or the I611S mutant <i>mmpL7</i> (<i>mmpL7^I611S</i>). Surface-exposed lipids (S) and lipids associated with the remaining cell pellet (P) were extracted and separated by TLC to resolve PDIM as in (A).</p>