The Identification and Location of Succinyl Residues and the Characterization of the Interior Arabinan Region Allow for a Model of the Complete Primary Structure of Mycobacterium tuberculosis Mycolyl Arabinogalactan

Abstract

The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-␣-D-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear ␣-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.The primary structure of the cell wall core of mycobacteria has been an object of study for many years. The fundamental concept that it consists of peptidoglycan attached to mycolic acids via the polysaccharide arabinogalactan (AG) 2 was determined some time ago (1). Also, early studies revealed a chemotype IV peptidoglycan similar to that of Escherichia coli with both diaminopimelic acid (DAP)-Ala and DAP-DAP crosslinking (2). Until very recently, little additional study of the peptidoglycan of M. tuberculosis has occurred. These recent studies have focused on substituents on the peptide carboxyl groups (3) and on whether the peptidoglycan chains are parallel or perpendicular to the plasma membrane (4). The structure of the mycolic acids was determined in detail some time ago (5); however, notably lacking in these studies was the structure of the arabinogalactan polymer, although the esterification of the mycolic acids at C5 of some arabinofuranosyl residues was established (6, 7). Also some linkage and ring form information on the arabinosyl and galactosyl residues was determined by methylation analysis (8 -10).The primary structure of AG is very complex and is not composed of a simple repeating unit as is the case for most bacterial polysaccharides. Elucidation of the structure was fundamentally advanced by the isolation and characterization of per-Oalkylated oligosaccharide alditols (11). This analysis revealed a linear galactan to which arabinan side chains were attached and also allowed elucidation of the detailed structure of the hexaarabinoside non-reducing end of the arabinan. The data were consistent with many different structures of the complete arabinan, and two possibilities were proposed for further study (11). During the same time period, the linker disaccharide (12) at the reducing end of AG and the exact location of the mycolic acids at the non-reducing end of the arabinan were determined (13). A follow-up study extended the structure of the arabinan at the non-reducing end from the hexa-arabinoside to include the last 17-22 arabinosyl residues (14), but until very recently, additional prog...

Identification of Mycobacterium tuberculosis Clinical Isolates with Altered Phagocytosis by Human Macrophages Due to a Truncated Lipoarabinomannan

“…In support of this concept, M. tuberculosis was recently found to contain a mammalian mannosyltransferase homologue (41), thus establishing that the process of mannosylation is conserved at least to some extent between M. tuberculosis and eukaryotic organisms. This concept of M. tuberculosis pathways mimicking aspects of human biosynthetic pathways is further supported by recent studies revealing other typical eukaryotic amino sugar substitutions on components of the M. tuberculosis cell envelope (42,43).…”

Detailed Structural and Quantitative Analysis Reveals the Spatial Organization of the Cell Walls of in Vivo Grown Mycobacterium leprae and in Vitro Grown Mycobacterium tuberculosis

“…M. tuberculosis (H37Rv) and M. smegmatis mc 2 155 were grown and harvested as described previously (16).…”

“…Additionally, the nonessentiality of the arabinogalactan biosynthetic genes in the closely related Corynebacterium glutamicum allowed definition of the nature of the attachment of the arabinan to the galactan (17). Finally, a recent report from our group has defined the entire primary structure of the cell wall mycolyl arabinogalactan identifying the location of the attached succinyl residues and re-defining the structure of the interior arabinan chain of M. tuberculosis and M. smegmatis (16).…”

“…Analyses of the O-alkylated alditols of AG-derived oligosaccharides revealed the following: (i) the attachment of arabinan side chains to a linear galactan chain, with a specific hexaarabinoside at its nonreducing end (12); (ii) the linker disaccharide (Rha-GlcNAc)-P at the reducing end (13) of the entire AG molecule; and (iii) the location of the outer membrane-located mycolyl esters at the nonreducing end of the arabinan (14). The use of a partially purified endo-arabinase from Mycobacterium smegmatis that specifically cleaves the ␣-1,5 linkages allowed released oligoarabinans to be characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF) (15,16) and the realization that two hexaarabinosides are connected to yield an Ara 17 unit (see Fig. 2).…”

Broadening Our View About the Role of Mycobacterium tuberculosis Cell Envelope Components During Infection: A Battle for Survival

“…Clusters of four mycolic acids are then attached to the terminal arabinofuranosyl motifs of non-reducing ends of the arabinan chain via ester linkage. Approximately two-thirds of the non-reducing ends of arabinan are mycolated at the 5 position of Araf residues (Daffe et al, 1993), one-third with succinyl and one-third with glucosaminosyl residues (Bhamidi et al, 2008). There are approximately 2-3 arabinan chains attached to the galactan core (Baulard et al, 1998;Brennan and Nikaido, 1995).…”