Functional analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative secretome analysis of a mycobacterial lgt mutant

Abstract

Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Using Lgt from Escherichia coli in a BLASTp search, we identified the corresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt in Mycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis ΔMSMEG_3222 mutant could be generated. Using Triton X-114 phase separation and [(14)C]palmitic acid incorporation, we demonstrate that MSMEG_3222 is the major Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell envelope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis ΔMSMEG_3222 strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including 20 lipoproteins. All lipoproteins were secreted at higher levels in the ΔMSMEG_3222 mutant. We identify the major Lgt in M. smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuberculosis lgt is essential and thus a validated drug target.

Fig 1

Disruption of M. smegmatis lgt (MSMEG_3222). (Left) Genomic DNAs from M. smegmatis (lane 1), lgt single-crossover (5′sco) mutant (lane 2), Δlgt mutant (lane 3), and Δlgt-lgt_Mtb mutant (lane 4; lgt_Mtb indicates lgt from M. tuberculosis) were digested with XcmI and probed with a 967-bp ApaI lgt gene fragment. The presence of a single 5.6-kbp fragment in the Δlgt knockout strain compared to the single 5.4-kbp fragment in the parental strain demonstrates inactivation of lgt. The shift in fragment size in the Δlgt knockout strain results from replacement of a 959-bp lgt fragment with a 1.2-kbp kanamycin resistance cassette. (Right) Genomic DNAs of M. smegmatis (lane 5), lgt-5′ single-crossover mutant (lane 6), Δlgt mutant (lane 7), and Δlgt-lgt_Mtb mutant (lane 8) were digested with BamHI and probed with a 491-bp SacI M. tuberculosis lgt gene fragment to demonstrate complementation. Complementation is indicated by a hybridization signal with genomic DNA derived from strain Δlgt-lgt_Mtb (the M. tuberculosis lgt probe does not hybridize with M. smegmatis lgt).

Fig 2

Growth characteristics of M. smegmatis Δlgt. (A) M. smegmatis wild type, M. smegmatis Δlgt, and M. smegmatis Δlgt-lgt_Mtb were grown in rich medium (a) or in nutrient-poor medium (b). (B) Inactivation of lgt affects cell and colony morphology. (a) Microcolonies of M. smegmatis grown on 7H10 agar supplemented with OADC for 3 days (total magnification, ×10); (b) auramine-stained M. smegmatis grown in 7H9-Tween (total magnification × 2000); (c) Ziehl-Neelsen-stained M. smegmatis grown in 7H9-Tween (total magnification, ×1,000); (d) electron microscopic photographs of M. smegmatis (total magnification, ×200,000). Column 1, parental M. smegmatis; column 2, Δlgt mutant; column 3, Δlgt-lgt_Mtb complemented strain; arrow, cytoplasmic membrane; square, electron-translucent layer; circle, electron-dense outer layer.

Fig 3

M. smegmatis Δlgt mutant fails to attach the lipid anchor. (A) Triton X-114 phase partition of M. smegmatis strains expressing recombinant lipoprotein Mpt83. (B) Triton X-114 phase partition of M. smegmatis parental strain, Δlgt mutant, and Δlgt-lgt_Mtb complemented strain expressing recombinant LppX using anti-LprG antibody. Of note, anti-LprG antibody (provided by H. Bercovier) cross-reacts with LppX (see Fig. S1 in the supplemental material). (C) [¹⁴C]palmitic acid incorporation in the wild type and Δlgt mutant.

Fig 4

Lipoproteins without a lipid anchor are not cleaved by LspA. Western blot analysis of whole-cell extracts of M. smegmatis strains expressing Mpt83 (A) or LppX (B). The signal peptide of lipoproteins Mpt83 and LppX accounts for approximately 2 kDa. The signal peptide is not cleaved in the lgt mutant in the case of LppX. The signal peptide of Mpt83 is cleaved in the Δlgt mutant, indicating Lgt-independent signal sequence cleavage. Western blot analyses were performed using anti-HA antibody and corresponding secondary antibody conjugated with horseradish peroxidase.

Fig 5

Subcellular localization of Mpt83 and LppX in M. smegmatis Δlgt mutant and parental strains. Western blot analyses of fractionated M. smegmatis extracts are shown. Lipoproteins Mpt83 and LppX localize in the cell wall fraction in the parental strain but are absent from the cytoplasmic membrane and the cell wall fraction in the Δlgt mutant. Absence of lipoproteins in the cell envelope fractions suggests that lipoproteins in the Δlgt mutant are released into the supernatant (see Fig. 6 in the supplemental material).

Fig 6

Close-ups of the lipoproteins that are secreted into the medium in the Δlgt mutant due to the missing lipid anchor. Shown are sections of the dual-channel images of the secretome of the M. smegmatis Δlgt mutant (red image) and the wild-type strain (green image) at the transition phase (t₀; A) and 1 h after entry into the stationary phase (t₁; B). The secretome was precipitated with TCA and separated using 2D PAGE in the pH range of 4 to 7, as described in Materials and Methods. Quantification of the dual-channel image was performed using Decodon Delta 2D software. (C) The induction ratios of the identified lipoproteins in the extracellular proteome of the lgt mutant to those of the wild type are shown in the corresponding diagram. Two biological replicates are used for quantification in panel C. Proteins are annotated with their corresponding MSMEG_ number. Lipoproteins were identified using LipoP 1.0.