Two Accessory Proteins Govern MmpL3 Mycolic Acid Transport in Mycobacteria

Abstract

Mycolic acids are the signature lipid of mycobacteria and constitute an important physical component of the cell wall, a target of mycobacterium-specific antibiotics and a mediator of Mycobacterium tuberculosis pathogenesis. Mycolic acids are synthesized in the cytoplasm and are thought to be transported to the cell wall as a trehalose ester by the MmpL3 transporter, an antibiotic target for M. tuberculosis However, the mechanism by which mycolate synthesis is coupled to transport, and the full MmpL3 transport machinery, is unknown. Here, we identify two new components of the MmpL3 transport machinery in mycobacteria. The protein encoded by MSMEG_0736/Rv0383c is essential for growth of Mycobacterium smegmatis and M. tuberculosis and is anchored to the cytoplasmic membrane, physically interacts with and colocalizes with MmpL3 in growing cells, and is required for trehalose monomycolate (TMM) transport to the cell wall. In light of these findings, we propose MSMEG_0736/Rv0383c be named "TMM transport factor A", TtfA. The protein encoded by MSMEG_5308 also interacts with the MmpL3 complex but is nonessential for growth or TMM transport. However, MSMEG_5308 accumulates with inhibition of MmpL3-mediated TMM transport and stabilizes the MmpL3/TtfA complex, indicating that it may stabilize the transport system during stress. These studies identify two new components of the mycobacterial mycolate transport machinery, an emerging antibiotic target in M. tuberculosisIMPORTANCE The cell envelope of Mycobacterium tuberculosis, the bacterium that causes the disease tuberculosis, is a complex structure composed of abundant lipids and glycolipids, including the signature lipid of these bacteria, mycolic acids. In this study, we identified two new components of the transport machinery that constructs this complex cell wall. These two accessory proteins are in a complex with the MmpL3 transporter. One of these proteins, TtfA, is required for mycolic acid transport and cell viability, whereas the other stabilizes the MmpL3 complex. These studies identify two new components of the essential cell envelope biosynthetic machinery in mycobacteria.

Keywords: Mycobacterium; Mycobacterium tuberculosis; cell envelope; transporters.

FIG 1

MmpL3 and MSMEG_0736 form a complex. (A) Silver-stained SDS-PAGE gel of elutions from GFP-Trap purifications using detergent solubilized membranes from M. smegmatis expressing MSMEG_0736-msfGFP (MGM6425) or MmpL3-msfGFP (MSMEG_0250 [MGM6464]). See Table 1 for protein identifications. (B) Silver-stained SDS-PAGE gel of the first elution from a GFP-Trap purification using detergent solubilized membranes from M. smegmatis expressing MSMEG_0736-msfGFP. The band corresponding to the molecular weight of MmpL3, indicated with an asterisk, was excised and subjected to mass spectrometry analysis and identified as MmpL3 (see Materials and Methods and Table S2 in the supplemental material).

FIG 2

MsTtfA/MtbTtfA is required for mycobacterial growth and cell elongation. (A) M. smegmatis strains with deletion of chromosomal ttfA and carrying a copy of ttfA at the attB phage integration site were subjected to marker exchange with attB integrating vectors. Δttfa attB::ttfA strep (MGM6414) strains transformed with pMV306kan (vector), pAJF792 (encoding MsTtfA), or pAJF793 (MtbTtfA) are shown on kanamycin agar. (B) Ten-fold dilutions of M. smegmatis carrying ATc-inducible CRISPRi nontargeting control (NT [MGM6418]) or ttfA (MGM6419) on agar media with and without ATc. (C) Growth curves of nontargeting (MGM6418, blue) and ttfA-targeting (MGM6419, red) CRISPRi M. smegmatis strains grown under uninduced (solid, closed circles) and ATc-induced (dashed, empty circles) conditions. (D) Growth curves of nontargeting (MGM6715, blue) or three distinct M. tuberculosis ttfA-targeting CRISPRi M. tuberculosis strains (MGM6675, red; MGM6677, green; MGM6679, purple) grown under uninduced (solid, closed circles) and ATc-induced (dashed, empty circles) conditions. (E) Fluorescence microscopy of an M. smegmatis ttfA-targeting CRISPRi strain marked with MalF(1,2)-mCitrine (MGM6433) 15 h post-CRISPRi induction with ATc (top, −TtfA) or, an uninduced control at 15 h (+TtfA, bottom). YFP (left) and DIC (right) images shown. Exposure times for YFP, 250 ms, 40% LED. (F) Loss of TtfA leads to short cells. Cell lengths of nontargeting (MGM6418, triangles) and TtfA-targeting (MGM6419, circles) CRISPRi strains induced for 12 h. Representative DIC/FM 4-64 images used for quantitation shown above the graph. Error bars are standard deviation and ** indicates P < 0.001.

FIG 3

TtfA is a membrane protein that localizes to poles and septa. (A) M. smegmatis TtfA-mCitrine expression strain (MGM6423) imaged during logarithmic growth. YFP (left), DIC (middle), and overlay (right) images shown. Bars, 1 μm. Exposure time for YFP, 1 s, 75% LED. (B) Localization of TtfA-msfGFP by cellular fractionation. Cell-free supernatant and cell pellet fractions (left) and soluble and membrane fractions (right) probed for secreted protein Ag85 (top), membrane protein FtsY (top middle), cytoplasmic protein RpoB (bottom middle), and GFP for TtfA-msfGFP (bottom).

FIG 4

TtfA and MmpL3 form a complex in vivo via the essential region of TtfA and independently of TMM synthesis. (A) DDM-solubilized M. smegmatis lysates (left) and GFP-Trap eluates (right) of msfGFP-expressing control (MGM6828) and TtfA-msfGFP (MGM6815) both coexpressing MmpL3-mCherry and probed with anti-RFP (top) and anti-GFP (bottom). (B) The essential region of MsTtfA is necessary and sufficient for MmpL3 interaction. DDM-solubilized lysates (top) and GFP-Trap eluates (bottom) of msfGFP control (MGM6828), full-length TtfA-msfGFP (1-278 [MGM6829]), or TtfA-msfGFP truncations (1-23 [MGM6826], 1-50 [MGM6823], 1-100 [MGM6827], 1-150 [MGM6824], 1-205 [MGM6822], 24-278 [MGM6825]) coexpressing MmpL3-mCherry and probed with anti-RFP (top) and anti-GFP (bottom). (C) The MsTtfA-MmpL3 interaction is independent of mycolate synthesis. GFP-Trap eluates of MmpL3-mCherry expression strains coexpressing msfGFP control (MGM6828) or TtfA-msfGFP with either control CRISPRi (NT [MGM6816]) or Pks13 depleted (MGM6817) for 6 h with ATc. Top panel is probed for MmpL3-mCherry with anti-RFP and bottom with anti-GFP.

FIG 5

TtfA and MmpL3 colocalize at cell poles and septa independently of TMM synthesis. (A) Localization of MsTtfA-mCherry/MmpL3-msfGFP (MGM6433, top) and MmpL3-mCherry/TtfA-msfGFP (MGM6434, bottom). (B) Localization of TtfA-msfGFP or MmpL3-msfGFP in Pks13-depleted or mock-depleted cells.

FIG 6

MsTtfA and MtbTtfA are required for TMM transport. (A) Thin-layer chromatographs (TLCs) of extractable mycolic acids from three replicate ¹⁴C-acetic acid-labeled M. smegmatis cultures carrying CRISPRi targeting guide RNAs (nontargeting [MGM6418]; left), ttfA (MGM6419; middle), or mmpL3 (MGM6637; right). Isoniazid treatment served as a positive control for inhibition of all mycolate synthesis. (B) Graph of TDM/TMM ratios for quantitation of TMM and TDM of TLCs in panel A. (C) TLCs of extractable mycolic acids from three replicate ¹⁴C-acetic acid-labeled M. tuberculosis cultures depleted for TtfA (MGM6675; left) or MmpL3 (MGM6676; right) (D) Quantitation of TDM/TMM ratios from quantitation of TMM and TDM of TLCs in panel C. ***, P < 0.01.

FIG 7

MSMEG_5308-msfGFP accumulates in response to MmpL3 dysfunction. (A) Lysates of MSMEG_5308-msfGFP expression strains with CRISPRi constructs nontargeting control (NT [MGM6766]), MmpL3 (MGM6718), TtfA (MGM6717), or Pks13 (MGM6767) (ATc induction at 0, 2, 4, 6, and 8 hrs) and probed with anti-GFP or anti-RpoB. Anti-GFP blot shown between 75 kDa and 63 kDa as determined by molecular weight marker. (B) Lysates of MSMEG_5308-msfGFP expression strain (MGM6681) treated with dimethyl sulfoxide [DMSO], 10 μg/ml INH, 20 μg/ml INH, 5 μM SQ109, 5 μM BM212, or 5 μM AU1235 for 0, 1.5, or 3 hrs and probed with anti-GFP and anti-RpoB (loading control).

FIG 8

MSMEG_5308 localizes to cell poles and septa and stabilizes the TtfA/MmpL3 interaction. (A) MSMEG_5308-msfGFP expression strain (MGM6681) imaged during logarithmic growth. GFP, DIC, and merged images are shown. (B) GFP-Trap pulldown of TtfA-msfGFP/MmpL3-mCherry coexpression strains with nontargeting or MSMEG_5308-targeting CRISPRi constructs. Inputs (left) and eluates (right) from the GFP-Trap column in the presence of either DDM or Triton X-100.