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 mycolylarabinogalactan-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 6,69-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.

Functional Analysis of the Holin-Like Proteins of Mycobacteriophage Ms6

“…In some phages, like and 21, the holin gene presents a dual-start motif producing two proteins by virtue of alternate translational starts, the holin and the antiholin, while in other phages these two proteins are encoded by separate genes (e.g., P1 and T4) (29,42). Such diversity is also observed in mycobacteriophages: in addition to the endolysin LysA, the majority of mycobacteriophages sequenced so far encodes an additional enzyme with lipolytic activity, LysB, that targets the outer membrane of mycobacteria (5,6). In phages belonging to cluster A2, like D29, the holin gene is positioned between lysA and lysB while in phages belonging to cluster F1, as in the Ms6 case, the holin gene is localized immediately downstream of lysB.…”

“…Even though the mechanisms underlying mycobacteriophage lysis of mycobacteria are poorly understood, recent work has contributed significantly to the progress in the field (2,5,6,14,25). Nonetheless, the exact mechanism by which the lysis effectors LysA and LysB are localized to their substrates remains elusive in the majority of the mycobacteriophages.…”

Genomic and Functional Analyses of Rhodococcus equi Phages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7

“…In addition to the cytoplasmic membrane and the peptidoglycan cell wall, the envelope of the Rhodococcus and the other genera of the mycolata, a suprageneric taxon including Nocardia, Corynebacterium, and Mycobacterium, has a third layer comprised of an arabinogalactan polymer linked to an "outer membrane" of mycolic acid chains (70). Extrapolating from the lysis systems of mycobacteriophages, rhodococcal phages can be expected to encode not only the canonical holin and endolysin (LysA) lysis proteins but also an enzyme, LysB, that attacks this outer layer (21,22,54). The LysA and holin genes were readily identified as adjacent reading frames, gene10 and gene11 in Pepy6, and as a .…”

Mycobacteriophage Ms6 LysA: a Peptidoglycan Amidase and a Useful Analytical Tool

“…Alternatively, the polycationic properties of the lysozyme (28) may contribute to the molecule's lethal effect on M. smegmatis; however, it is known that relatively minor changes in the mycolic acid structure result in a dramatic increase in the lysozyme susceptibility of Mycobacterium marinum (29), suggesting that minor changes in the physical nature of the mycolic acid layer can result in significant changes in the permeability of this hydrophobic barrier. Recently, an intriguing idea that mycobacterial pathogens might be rendered susceptible to exogenous endolysins through cotreatment with LysA and LysB, a mycobacteriophage mycolylarabinogalactan esterase that releases mycolic acids from the mycobacterial cell wall, was proposed (17,30). Utilization of LysA for peptidoglycan analysis.…”