Polymerization of Mycobacterial Arabinogalactan and Ligation to Peptidoglycan

Abstract

The cell wall of Mycobacterium spp. consists predominately of arabinogalactan chains linked at the reducing ends to peptidoglycan via a P-GlcNAc-(␣1-3)-Rha linkage unit (LU) and esterified to a variety of mycolic acids at the nonreducing ends. Several aspects of the biosynthesis of this complex have been defined, including the initial formation of the LU on a polyprenyl phosphate (Pol-P) molecule followed by the sequential addition of galactofuranosyl (Galf) units to generate Pol-P-P-LU-(Galf) 1,2,3, etc. and Pol-P-P-LU-galactan, catalyzed by a bifunctional galactosyltransferase (Rv3808c) capable of adding alternating 5-and 6-linked Galf units. By applying cell-free extracts of Mycobacterium smegmatis, containing cell wall and membrane fragments, and differential labeling with UDP-[ 14 C]Galp and recombinant UDP-Galp mutase as the source of [ C]Galf for galactan biosynthesis and 5-P-[14 C]ribosyl-P-P as a donor of [ 14 C]Araf for arabinan synthesis, we now demonstrate sequential synthesis of the simpler Pol-P-P-LU-(Galf) n glycolipid intermediates followed by the Pol-P-P-LU-arabinogalactan and, finally, ligation of the P-LU-arabinogalactan to peptidoglycan. This first time demonstration of in vitro ligation of newly synthesized P-LUarabinogalactan to newly synthesized peptidoglycan is a necessary forerunner to defining the genetics and enzymology of cell wall polymer-peptidoglycan ligation in Mycobacterium spp. and examining this step as a target for new antibacterial drugs.The cell envelope of Mycobacterium tuberculosis is composed of a conventional plasma membrane and a cell wall proper unique to some genera within the Actinomycetales order, consisting of a core of arabinogalactan (AG), 1 peptidoglycan (PG), and mycolic acids interspersed with a variety of free lipids, lipoglycans, and proteins (1); there is also evidence for polysaccharides on the outer face of the cell wall (2). The mycolic acids are attached to the nonreducing ends of the arabinogalactan, whereas the reducing ends are covalently attached to the crosslinked peptidoglycan via phosphoryl-N-acetylglucosaminosylrhamnosyl linkage units (P-GlcNAc-Rha). This massive structure, the mycolate-arabinogalactan-peptidoglycan-complex (MAPc), is the basis of many of the physiological and pathogenic features of M. tuberculosis and the site of susceptibility and resistance to many of the anti-tuberculosis drugs (3). Biosynthesis of this complex commences with attachment of the residues of the linkage unit, GlcNAc-1-P and Rha, donated by UDP-GlcNAc and dTDP-Rha, respectively, to a polyprenyl phosphate (Pol-P) carrier lipid (4). Formation of the linkage unit is followed by the sequential addition of galactofuranosyl (Galf) units donated by UDP-Galf, to provide simple Pol-P-Plinked AG intermediates (4). The bulk, if not all, of galactan biosynthesis is catalyzed by a membrane-associated bifunctional galactosyltransferase capable of adding the alternating 5-and 6-linked Galf units (5, 6).The demonstration that the direct donor of the arabinofuranosyl (Araf) unit...

Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate

“…UDP-MurNAc was enzymatically synthesized from UDP-GlcNAc by use of recombinant MurA and MurB of E. coli as previously described (20). UDP-MurNAc-L-Ala-D-Glu-DAP-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) was synthesized enzymatically (28,42) smegmatis. For the synthesis of radiolabeled MurNAc-(pentapeptide)-diphosphoryl-prenol (lipid I), a particulate enzyme fraction, containing cell wall, membrane, and small amounts of cytosol, from M. smegmatis was prepared as previously described (26) and preincubated with 50 g of ramoplanin/ml for 10 min, For large-scale, in vitro synthesis of lipid I, 2 g of protein of M. smegmatis particulate enzyme was resuspended in 400 ml of buffer A containing 50 g of ramoplanin/ml and incubated for 10 min at 28°C followed by the addition of ATP and UDP-MurNAc-pentapeptide to final concentrations of 100 and 50 M, respectively.…”

Deletion of Cg-emb in Corynebacterianeae Leads to a Novel Truncated Cell Wall Arabinogalactan, whereas Inactivation of Cg-ubiA Results in an Arabinan-deficient Mutant with a Cell Wall Galactan Core

“…Chemical analysis of the mature lipidlinked galactan, synthesized in vitro (11), suggests that this intermediate then serves as the acceptor for the subsequent addition of arabinofuranose (Araf) residues from the arabinose sugar donor ␤-D-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) in the formation of the Araf portion (␣135, ␣133, and ␤132 linkages) of AG (15)(16)(17)(18). The AG-lipid intermediate at some point is mycolylated and transglycosylated to peptidoglycan (19,20).…”

Identification of the namH Gene, Encoding the Hydroxylase Responsible for the N-Glycolylation of the Mycobacterial Peptidoglycan

“…The arabinogalactan is in turn covalently attached to the peptidoglycan via an essential rhamnose-Nacetylglucosamine linker, forming the mycolylarabinogalactanpeptidoglycan complex (mAGP) (3,4). The peptidoglycan ultimately serves to anchor the principle mass of the mycobacterial cell envelope.…”