The MmpL3 interactome reveals a complex crosstalk between cell envelope biosynthesis and cell elongation and division in mycobacteria

Abstract

Integral membrane transporters of the Mycobacterial Membrane Protein Large (MmpL) family and their interactome play important roles in the synthesis and export of mycobacterial outer membrane lipids. Despite the current interest in the mycolic acid transporter, MmpL3, from the perspective of drug discovery, the nature and biological significance of its interactome remain largely unknown. We here report on a genome-wide screening by two-hybrid system for MmpL3 binding partners. While a surprisingly low number of proteins involved in mycolic acid biosynthesis was found to interact with MmpL3, numerous enzymes and transporters participating in the biogenesis of peptidoglycan, arabinogalactan and lipoglycans, and the cell division regulatory protein, CrgA, were identified among the hits. Surface plasmon resonance and co-immunoprecipitation independently confirmed physical interactions for three proteins in vitro and/or in vivo. Results are in line with the focal localization of MmpL3 at the poles and septum of actively-growing bacilli where the synthesis of all major constituents of the cell wall core are known to occur, and are further suggestive of a role for MmpL3 in the coordination of new cell wall deposition during cell septation and elongation. This novel aspect of the physiology of MmpL3 may contribute to the extreme vulnerability and high therapeutic potential of this transporter.

Figure 1

The M. tuberculosis mycolic acid biosynthetic pathway. The C48–C54 meromycolate chain is biosynthesized by FAS-II through the processive addition of multiple malonate units onto C16–C26 precursors generated by FAS-I. The initial substrates of FAS-II are thus medium length keto-acyl-ACP resulting from the condensation by the M. tuberculosis FabH protein of the acyl-CoA products of FAS-I with malonyl-ACP. After reduction by the β-keto-acyl-ACP reductase MabA, dehydration by the (3R)-hydroxyacyl dehydratases HadAB and HadBC, and reduction by the enoyl-CoA reductase InhA, either the β-ketoacyl-ACP synthase KasA or KasB catalyzes the condensation of the resulting product with malonyl-ACP units, thereby initiating the next round of elongation. The products of FAS-II may undergo further elongation and functional modifications of the meromycolic acid chain, catalyzed in part by S-adenosyl methionine-dependent methyltransferases (MmaA1, MmaA2, MmaA3, MmaA4, CmaA1, CmaA2, PcaA, UmaA), prior to the Pks13/FadD32-mediated condensation of the activated α-branch with the meromycolic acid chain to yield the full-size mycolic β-ketoester. Pks13 transfers mycolic β-ketoester to trehalose prior to reduction by the CmrA reductase yielding mature TMM. TmaT acetylates the mycolic acid in TMM prior to its export by MmpL3 and other potential components of a translocation machinery spanning the mycobacterial cell envelope. The mycolyltransferases encoded by fbpA, fbpB and fbpC catalyze the transfer of mycolic acids from TMM to arabinogalactan (AG), or to another TMM molecule generating TDM. Proteins in red font were tested individually for interaction with MmpL3 by BATCH two-hybrid system.

Figure 2

mmpL3 genomic region in M. tuberculosis, M. smegmatis and M. leprae. Conserved genes are in yellow and were all tested for interaction with MmpL3 by BATCH two-hybrid system. Non-conserved genes (in grey) and pseudogenes (bold lines) were not tested. mmpL3 and orthologs are in green.

Figure 3

Quantification of in vivo interactions between full-size or C-terminal truncated MmpL3 and protein partners. MmpL3 (full-size) and MmpL3^1–744 fusion proteins harboring C-terminal or N-terminal T18 and T25 domains were generated as “baits” and systematically compared for pairwise interactions with the binding partners listed in Table 1. Pairwise co-transformants developing a blue color in the BATCH screen were grown in LB broth, and the cultures processed for β-galactosidase activity as described in Methods. The values presented are the mean activities (relative units) ± standard error from measurements performed on three independent E. coli transformants.

Figure 4

Quantification of in vivo interactions between MmpL3 binding partners. CrgA, Wag31, TmaT, AftD, Rv0227c, Rv0204c and MmpL11 fusion proteins harboring C-terminal or N-terminal T18 and T25 domains were generated as “baits” and tested for pairwise interactions with other MmpL3 binding partners. Pairwise co-transformants developing a blue color in the BATCH screen were grown in LB broth, and the cultures processed for β-galactosidase activity as in Fig. 3.

Figure 5

Quantification of in vitro interactions between MmpL3 and binding partners by surface plasmon resonance. SPR was used to analyze kinetics of the purified MmpL3 interaction with various proteins. Binding sensorgrams were collected by injecting two-fold increasing concentrations ranging from 0.6 μM up to 10 μM of AftD (violet), Rv0207c (blue) and CrgA (green). Binding curves were fitted globally into a simple 1:1 model (red lines) and the dissociations constants are shown for each protein. No specific binding was detected for the Ag85 complex, Pks13, MviN and LprC [see Fig. S3].

Figure 6

CrgA interacts with MmpL3 in intact mycobacterial cells. Left: In-gel fluorescence of DSP-treated, detergent-solubilized, MmpL3tb-protein complexes prepared from MsmgΔmmpL3/pMVGH1-mmpL3tb-gfp + pFAX-crgA cells reveals the presence of high molecular weight MmpL3-GFP protein complexes in the elution fractions that are reduced upon addition of DTT. The expected size of MmpL3-GFP is ~126 KDa. Right: Immunoblot analysis of DSP-treated, detergent-solubilized, MmpL3tb-protein complexes prepared from Msmg + pFAX-crgA and MsmgΔmmpL3/pMVGH1-mmpL3tb-gfp + pFAX-crgA cells. The immunoblot shows the presence of CrgA-FLAG in the elution fractions from MsmgΔmmpL3/pMVGH1-mmpL3tb-gfp + pFAX-crgA cells but not in those from cells devoid of mmpL3tb-gfp expression plasmid. The expected size of CrgA-FLAG is ~12 KDa. *Denotes a non-specific M. smegmatis protein reacting with the anti-FLAG antibody. The full-length gel and blots are shown. Co-affinity purifications were performed twice on independent culture batches with the same results.

Figure 7

Loss of polar localization of MmpL3 upon truncation of the C-terminal domain. Representative fluorescence images of MsmgΔmmpL3/pMVGH1-mmpL3tb-gfp (top panels) and MsmgΔmmpL3/pMVGH1-mmpL3tb^1–744-gfp (lower panels) showing the focal localization of full-size MmpL3tb-GFP at the old poles and septa of actively dividing bacilli, and dimmer and more diffuse fluorescent signal corresponding to the truncated form of the transporter. The fluorescence intensity of 50 bacilli from each strain (red line) in one representative experiment, plus or minus standard deviation (black lines), was scored and plotted against normalized cell length (right panels).

Figure 8

Network of M. tuberculosis proteins found to interact with MmpL3. Blue lines indicate protein interactions established in the context of the present study. Protein-protein interactions established in the context of previous studies are connected by red lines^,,. The functional groups to which the proteins belong or are thought to belong are color-coded. Proteins in yellow circles are related to mycolic acid metabolism. Proteins in the orange circle have not yet been assigned a function. LAM, lipoarabinomannan; LM, lipomannan; AG, arabinogalactan.