Novel Polyoxyethylene-Containing Glycolipids Are Synthesized in Corynebacterium matruchotii and Mycobacterium smegmatis Cultured in the Presence of Tween 80

Abstract

The addition of polyoxyethylene sorbitan monooleate (Tween 80) to a culture of mycobacteria greatly influences cell permeability and sensitivity to antibiotics but very little is known regarding the underlying mechanism. Here we show that Corynebacterium matruchotii (surrogate of mycobacteria) converts Tween 80 to a structural series of polyoxyethylenic acids which are then used to form novel series-2A and series-2B glycolipids. Minor series-3 glycolipids were also synthesized. The polyoxyethylenic acids replaced corynomycolic acids in the cell wall. Correspondingly the trehalose dicorynomycolate content was reduced. MALDI mass spectrometry, MS-MS, (1)H-NMR, and (13)C-NMR were used to characterize the series-2 glycolipids. Series-2A glycolipid is trehalose 6-C(36:2)-corynomycolate-6'-polyoxyethylenate and series-2B glycolipid is trehalose 6-C(36:2)-corynomycolate-6'-furan ring-containing polyoxyethylenate. Mycobacterium smegmatis grown in the presence of Tween 80 also synthesizes series-2 type glycolipids. The synthesis of these novel glycolipids in corynebacteria and mycobacteria should result in gross changes in the cell wall permeability and drug sensitivity.

Figure 1

Partial MALDI mass spectrum of TDCM-like glycolipid from C. matruchotii grown in the presence of Tween 80. The glycolipids in the chloroform/methanol extract were purified by silica gel and Sephadex LH20 column chromatography. Molecular ion peaks (MNa⁺) at m/z 613 to 921 represent series-3 glycolipids and those at m/z 997 to 1595 represent series-2 glycolipids. TDCM (m/z 1425) represents a glycolipid containing two C_36:2-corynomycolates.

Figure 2

MALDI mass spectrum of methyl polyoxyethylenates derived from purified series-2 glycolipid from C. matruchotii grown in the presence of Tween 80. The free acids from TBAH-hydrolyzed series-2 glycolipid were esterified with methyl iodide and analyzed by MALDI mass spectrometry.

Figure 3

Predicted products of degradation of Tween 80 by C. matruchotii. Structure A is polyoxyethylenic acid derived from the furan ring of Tween 80 (http://www.sigma-aldrich.com/) and structure B is the furan ring-associated polyoxyethylenic acid. Structures C and D are further degradation products of B. The terminal hydroxyl group is thought to be oxidized to the carboxyl group and all of these products are used by the bacteria to synthesize series-2A and -2B glycolipids. Alternative to structure B, the carboxylic acid group could be on the other polyoxyethylenate unit. The proposed structures of series-2A and -2B glycolipids are shown in upper right and lower right panels, respectively.

Figure 4

MS-MS spectrum of the lifted MNa⁺ peak at m/z 1111. Inset shows the structure and fragmentation pattern of this glycolipid from series-2B mass spectrum (Figure S1).

Figure 5

MS-MS spectrum of the lifted MNa⁺ peak at m/z 1331. Inset shows the structure and fragmentation pattern of this glycolipid from series-2B mass spectrum (Figure S1).

Figure 6

MALDI mass spectrum of decanoyl polyoxyethylenates derived from the cell wall of C. matruchotii grown in the presence of Tween 80. The free acids from TBAH-hydrolyzed cell wall skeleton were esterified with 1-iododecane, purified by silica gel column chromatography and analyzed by MALDI mass spectrometry. Three degraded series of decanoyl polyoxyethylenates are A, B, and C.

Figure 7

MALDI mass spectrum of series-2-type glycolipid in the purified “TDM-like” lipid fraction of M. smegmatis grown in the presence of Tween 80. 2A and 2B represent two structural series of polyoxyethylenate-containing glycolipids.