Mycobacteriophage Ms6 LysB specifically targets the outer membrane of Mycobacterium smegmatis

Abstract

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan-peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6'-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.

Fig. 1.

LC-MS (negative ion mode) identification of mycolic acids released from mAGP after LysB treatment. (a) Total ion chromatograms from non-treated and treated (LysB) mAGP samples. (b) The negative ion mass spectra (averaged from 19 to 25 min) of non-treated and treated mAGP samples from m/z 920 to 1232. The mAGP sample treated with LysB showed the presence of several peaks, identified as mycolic acids. α, α-mycolates; α′, α′-mycolates; e, epoxy mycolates. The ion at m/z 980 corresponds to a calibration reference compound.

Fig. 2.

Thin layer chromatogram developed with chloroform/methanol/water (20 : 4 : 0.5 by vol.) of total lipid extract from M. smegmatis (lane 1), total lipid extract from M. smegmatis treated with LysB (2), Mtb TDM standard (3), total lipid extract from M. tuberculosis H37Ra (4) and total lipid extract from M. tuberculosis H37Ra treated with LysB (5).

Fig. 3.

Analysis of MAMEs by TLC and MS. (a) TLC fractionation of MAMEs into subclasses: thin-layer chromatogram of MAMEs from whole M. smegmatis (lane 1) and Msmeg_1 (lane 2). The development solvent was n-hexane/ethyl acetate (95 : 5, v/v), used three times. α, α-mycolates; α′, α′-mycolates; e, epoxy mycolates. (b) LC-MS analyses of MAME subclasses extracted from Msmeg_1, carried out in positive ion mode. Top, α-mycolates; middle, α′-mycolates; bottom, epoxy mycolates. The presence or absence of peaks marked with an asterisk (*) was variable and, based on their masses, was probably due to oxidation reactions occurring on the silica gel.

Fig. 4.

LC-MS analysis (positive ion mode) of purified Msmeg_1 treated with LysB and non-LysB-treated enzyme-treated Msmeg_1. The non-treated Msmeg_1 spectrum revealed the presence of several ion clusters between m/z 2158 and 2647. The isotope peaks containing ¹²C exclusively are labelled.

Fig. 5.

LC-MS (negative mode) identification of mycolic acids from released Msmeg_1 after LysB treatment (top panel). The bottom panel is a control with no LysB treatment. The mycolates, thus identified, were consistent with the more detailed analysis of TDM shown in Fig. 3. α, α-mycolates; α′, α′-mycolates; e, epoxy mycolates. The ion at m/z 980 is from the calibration standard; the ions at m/z 955, 996 and 1024 (found in both the control and the sample) are of unknown origin, and can also be seen in Fig. 1.