Enzymatic hydrolysis of trehalose dimycolate releases free mycolic acids during mycobacterial growth in biofilms

Abstract

Mycobacterial species, like other microbes, spontaneously form multicellular drug-tolerant biofilms when grown in vitro in detergent-free liquid media. The structure of Mycobacterium tuberculosis biofilms is formed through genetically programmed pathways and is built upon a large abundance of novel extracellular free mycolic acids (FM), although the mechanism of FM synthesis remained unclear. Here we show that the FM in Mycobacterium smegmatis biofilms is produced through the enzymatic release from constitutively present mycolyl derivatives. One of the precursors for FM is newly synthesized trehalose dimycolate (TDM), which is cleaved by a novel TDM-specific serine esterase, Msmeg_1529. Disruption of Msmeg_1529 leads to undetectable hydrolytic activity, reduced levels of FM in the mutant, and retarded biofilm growth. Furthermore, enzymatic hydrolysis of TDM remains conserved in M. tuberculosis, suggesting the presence of a TDM-specific esterase in this pathogen. Overall, this study provides the first evidence for an enzymatic release of free mycolic acids from cell envelope mycolates during mycobacterial growth.

FIGURE 1.

Regulated synthesis of FM in M. smegmatis biofilms. A, two-dimensional radio-thin layer chromatography of ¹⁴C apolar and polar lipids equivalent to 50,000 cpm extracted from planktonic and biofilm cultures of M. smegmatis is shown. The TLCs were developed in solvent (Sol.) systems A, C, and D as described previously (37). The lipids with significantly different level of ¹⁴C incorporation in the two cultures are annotated as Spot 1, Spot 2, and TG. The direction of migration in each dimension is marked at the left bottom. B, radio-TLC of ¹⁴C apolar lipids equivalent to 50,000 cpm extracted from 2-, 3-, and 4-day (d) biofilms of M. smegmatis, mc²155 wild-type (wt), ΔgroEL1 (Δg), and ΔgroEL1 complemented with pMsgroEL1 (comp) is shown. The arrow denotes FM. The quantity of FM is normalized to the 2-day biofilms of wild-type M. smegmatis. C, shown is exposure of 2-, 3-, and 4-day biofilms to 400 μg/ml rifampicin. Percentage survival was calculated using untreated culture at the respective stage as a reference.

FIGURE 2.

FM is released from TDM in the presence of mycobacterial lysates. A, radio-TLC of the lipids in reaction mixtures of ¹⁴C-labeled M. smegmatis planktonic lysate with either lysates of M. smegmatis biofilms (bf), heat-inactivated biofilm lysates (heat bf), or with BSA is shown. B, radio-TLC of the lipids in the reaction mixture of purified ¹⁴C-labeled cell wall (MsCW) with buffer containing BSA, biofilm lysate (bf), heat-inactivated biofilms lysate (bf heat), and planktonic lysate (plnk) is shown. C, radio-TLC of the reaction mixture as described in panel B is shown except that ¹⁴C-MsTDM was used instead of MsCW as a substrate. D, radio-TLC shows lipids in the reaction mixture of purified ¹⁴C-MsTDM with M. tuberculosis biofilms lysate (bf) and buffer containing BSA. Purified FM was used as a control in panels A–D. TLCs in panel A–D were developed in chloroform:methanol 96:4.

FIGURE 3.

Decreased synthesis of TDM in maturing biofilms. A, shown is radio-TLC of ¹⁴C apolar lipids corresponding to 50,000 cpm from 2-, 3-, and 4-day (d) biofilms of M. smegmatis, mc²155 wild-type (wt), and mc²155-ΔgroEL1 extracted in petroleum ether. The plot shows relative amount of spots corresponding to [¹⁴C]TDM using 2-day biofilms of wild type as reference. B, the total lipid content in each sample of panel A was visualized by charring the plate with 10%H₂SO₄ in ethanol. A cold purified TDM was used as a control (ctrl). The position of TDM is marked by arrow. The plot shows the relative amount of total TDM using 2-day biofilms of wild type as reference. C, TLC of Spot 2, originally identified with differential ¹⁴C incorporation in planktonic and biofilm cultures (Fig. 1 panel A), along with purified M. tuberculosis TDM (Mtb TDM) and M. smegmatis (MsTDM) is shown. D, shown is TLC of total apolar lipids equivalent to 50,000 cpm from ¹⁴C-labeled planktonic (plnk) and 4-day biofilm (bf) cultures seen after charring as described above. A cold purified MsTDM was used as a control. The TLCs were developed in chloroform:methanol:water (90:10:1).

FIGURE 4.

Defective biofilms of mc²155:fbpA mutant. A, 5-day biofilms of M. smegmatis, mc²155 wild-type (wt), mc²155:ΔfbpA, and mc²155:ΔfbpA:pMs6398 (complemented) strains are shown. B, shown is radio-TLC, developed in chloroform:methanol:water (90:10:1), of apolar lipids from M. smegmatis wild type (wt) and ΔfbpA mutant showing incorporation of ¹⁴C into MsTDM. The plot shows the relative amount of MsTDM from the two strains. C, shown is radio-TLC of the apolar lipids from the wild-type and mutant strains developed in chloroform:methanol (96:4) showing ¹⁴C incorporation into FM. The plot shows the relative amount of FM in the two strains. 5-Day biofilms of the strains were labeled for 6 h with [¹⁴C]glycerol, and total apolar lipids equivalent to 50,000 cpm from each sample were loaded and compared with either purified MsTDM in panel B (marked with an arrow) or purified FM in panel C.

FIGURE 5.

Msmeg_1529 is a TDM-specific hydrolase. A, multiple sequence alignment of serine esterases from M. smegmatis by ClustalX is shown. The conserved catalytic triad of GXSXG, Asp, and His are annotated. B, shown is radio-TLC of apolar lipids extracted in petroleum ether from a reaction mixture containing purified ¹⁴C-MsTDM lysates of recombinant E. coli expressing putative M. smegmatis serine esterase identified in panel A. The recombinant strains expressed the ORFs (marked below each lane) under isopropyl 1-thio-β-d-galactopyranoside-inducible LacI-dependent expression system. Reactions with BSA and recombinant E. coli containing empty vector (pET21b) were used as negative control. C, SDS-PAGE shows TDM hydrolase (TDMH) (Msmeg_1529) purified from recombinant strain used in panel B. D, shown is radio-TLC of lipids extracted from the reaction mixture of pure ¹⁴C-MsTDM with either TDM hydrolase or LysB from mycobacteriophage D29. E, radio-TLC of lipids extracted from reaction mixture containing pure [¹⁴C]mAGP with either TDM hydrolase or LysB. F, shown is radio-TLC of lipids extracted from a reaction mixture containing pure ¹⁴C-MtTDM (TDM from M. tuberculosis) with TDM hydrolase. Buffer with BSA as a negative control and pure FM as a reference was used in panels D, E, and F.

FIGURE 6.

Phenotype of ΔMsmeg_1529 mutant. A, radio-TLC shows the petroleum ether-extracted ¹⁴C lipids from the reaction mixture containing [¹⁴C]TDM and lysates of either wild-type (wt) or ΔMsmeg_1529. BSA was used as a negative control. Purified FM (marked with an arrow) was used as a reference. B, 3- and 4-day biofilms of mc²155 (wild type), mc²155:ΔMsmeg_1529, and mc²155:ΔMsmeg_1529 complemented with pMsmeg_1529 are shown. C, shown is radio-TLC of ¹⁴C-labeled apolar lipids equivalent to 50,000 cpm made at the 4-day stage of the three strains described in panel B. Lipids were labeled with [¹⁴C]acetate for 6 h before petroleum ether extraction. FM and TG are annotated. D, radio-TLC shows the petroleum ether-extracted ¹⁴C lipids from the reaction mixture containing ¹⁴C-labeled planktonic (plnk) lysate and lysate from either wild-type or ΔMsmeg_1529-, or ΔMsmeg_1529-complemented strains. BSA in the buffer was used as a negative control, and purified FM from M. smegmatis (marked with an arrow) was used as a reference.