Identification of Residues in MmpL7 Domain 2 Required for PpsE Interaction

<div><p>(A) Twelve MmpL7 domain 2 mutants defective for PpsE binding were isolated in a reverse two-hybrid screen. These amino acid substitutions are displayed on a linear map of MmpL7 domain 2, with changes to amino acids other than proline or glycine in bold. Amino acid numbers correspond to positions in full-length MmpL7. TM domains 7 and 8 are denoted by hatched bars.</p><p>(B) Yeast strains expressing the PpsE prey construct and various MmpL7 domain 2 bait plasmids were transferred onto X-gal indicator plates (inset), and reporter activity was quantified from liquid cultures by monitoring β-galactosidase activity.</p><p>(C) Beads containing equal amounts of MmpL7 domain 2 and the I611S mutant were incubated with protein extracts containing myc-tagged PpsE and washed. Bound proteins were eluted and separated by SDS-PAGE, and PpsE was visualized by Western blot using anti-myc antibodies. GST-coated beads served as a negative control, and 1% of the protein extract added to the pulldown was loaded as a positive control (“input”).</p></div

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></div

Dominant Negative Effect of MmpL7 Domain 2 Requires the Presence of Full-Length MmpL7

<div><p>(A) Wild-type cells, an <i>mmpL7</i> transposon mutant (<i>mmpL7⁻</i>), and a complete <i>mmpL7</i> knockout (Δ<i>mmpL7</i>) carrying either no plasmid (−) or the MmpL7 domain 2 expression construct (+). Labeled lipids were extracted from pellets as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010002#ppat-0010002-g004" target="_blank">Figure 4</a> and separated by TLC to resolve PDIM.</p><p>(B) Western blot of MmpL7 domain 2 showing that it is expressed at equivalent levels in the different <i>M. tuberculosis</i> strains.</p></div

MmpL7 Domain 2 Interacts with the PDIM Synthesis Enzyme PpsE

<div><p>(A) Yeast two-hybrid reporter strains harboring the indicated bait and prey plasmids were streaked onto solid media with or without leucine. Growth on leucine-negative plates indicates a positive interaction. MmpL7^d2, MmpL7 domain 2; MmpL8^d2, MmpL8 domain 2.</p><p>(B) The same strains as in (A) were transferred onto X-gal-containing indicator plates (inset), and reporter activity was quantified from liquid cultures using β-galactosidase assays.</p><p>(C) Linear representation of full-length PpsE protein (1,488 amino acids) with the MmpL7 interaction region denoted. Catalytic domains of PpsE are also shown. ACP, acyl carrier protein; AT, acyl transferase; CE, condensing enzyme; KS, ketoacyl synthase.</p></div

Model of PDIM Synthesis and Transport

<p>MmpL7 interacts with PpsE, a subunit of the Pps enzyme required for PDIM synthesis. We propose that MmpL7 acts as a scaffold to recruit PDIM synthesis machinery, including Pps and perhaps Mas, leading to coordinate synthesis and transport of PDIM across the cytoplasmic membrane (CM). Whether MmpL7, or other factors, are required for delivery of PDIM through the peptidoglycan (PG) and mycolyl-arabinogalactan (mAG) layers is unclear.</p

PDIM Synthesis and Export Pathway and Topology of MmpL7

<div><p>(A) Schematic of the known steps in the PDIM synthesis and transport pathway. PpsA–E and Mas are polyketide synthases that extend fatty acids to phthiocerol and mycocerosic acids, respectively [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010002#ppat-0010002-b16" target="_blank">16</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010002#ppat-0010002-b17" target="_blank">17</a>]. These are then esterified to produce PDIM. MmpL7 and DrrC are required for the transport of PDIM to the cell surface [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010002#ppat-0010002-b08" target="_blank">8</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0010002#ppat-0010002-b10" target="_blank">10</a>]. R is =O (keto) or –OCH₃ (methoxy).</p><p>(B) Predicted membrane topology of MmpL7 indicating the two non-TM domains 1 and 2.</p></div

Sulfolipid-1 Biosynthesis Restricts <i>Mycobacterium tuberculosis</i> Growth in Human Macrophages

<i>Mycobacterium tuberculosis</i> (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host–pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a <i>stf0</i>-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide <i>in vitro</i>, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the <i>stf0</i> mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages