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...

Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.

UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor of peptidoglycan and the rhamnose-GlcNAc linker region of mycobacterial cell wall. In Mycobacterium tuberculosis H37Rv genome, Rv1018c shows strong homology to the GlmU protein involved in the formation of UDPGlcNAc from other bacteria. GlmU is a bifunctional enzyme that catalyzes two sequential steps in UDP-GlcNAc biosynthesis. Glucosamine-1-phosphate acetyl transferase catalyzes the formation of N-acetylglucosamine-1-phosphate, and N-acetylglucosamine-1-phosphate uridylyltransferase catalyzes the formation of UDP-GlcNAc. Since inhibition of peptidoglycan synthesis often results in cell lysis, M. tuberculosis GlmU is a potential anti-tuberculosis drug target. In this study we cloned M. tuberculosis Rv1018c (glmU gene) and expressed soluble GlmU protein in E. coli BL21(DE3). Enzymatic assays showed that M. tuberculosis GlmU protein exhibits both glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridylyltransferase activities. We also investigated the effect on Mycobacterium smegmatis when the activity of GlmU is fully removed or reduced via a genetic approach. The results showed that activity of GlmU is required for growth of M. smegmatis as the bacteria did not grow in the absence of active GlmU enzyme. As the amount of functional GlmU enzyme was gradually reduced in a temperature shift experiment, the M. smegmatis cells became non-viable and their morphology changed from a normal rod shape to stubbyrounded morphology and in some cases they lysed. Finally a microtiter plate based assay for GlmU activity with an OD 340 read out was developed. These studies therefore support the further development of M. tuberculosis GlmU enzyme as a target for new anti-tuberculosis drugs.

The cell wall of mycobacteria consists of an outer membrane, analogous to that of Gram-negative bacteria, attached to the peptidoglycan (PG) via a connecting polysaccharide arabinogalactan (AG). Although the primary structure of these components is fairly well deciphered, issues such as the coverage of the PG layer by covalently attached mycolates in the outer membrane and the spatial details of the mycolic acid attachment to the arabinan have remained unknown. It is also not understood how these components work together to lead to the classical acid-fast staining of mycobacteria. Because the majority of Mycobacterium tuberculosis bacteria in established experimental animal infections are acid-fast negative, clearly cell wall changes are occurring. To address both the spatial properties of mycobacterial cell walls and to begin to study the differences between bacteria grown in animals and cultures, the cell walls of Mycobacterium leprae grown in armadillos was characterized and compared with that of M. tuberculosis grown in culture. Most fundamentally, it was determined that the cell wall of M. leprae contained significantly more mycolic acids attached to PG than that of in vitro grown M. tuberculosis (mycolate:PG ratios of 21:10 versus 16:10, respectively). In keeping with this difference, more arabinogalactan (AG) molecules, linking the mycolic acids to PG, were found. Differences in the structures of the AG were also found; the AG of M. leprae is smaller than that of M. tuberculosis, although the same basic structural motifs are retained.The hallmark of mycobacteria is their cell wall consisting of a peptidoglycan layer attached to a mycolic acid containing outer membrane via the polysaccharide arabinogalactan. Profound and fundamental questions remain about this architecture, including how division occurs and how molecules, both nutrients and drugs, enter the cell. Although its impermeability is stressed (1), anomalies exist such as the susceptibility of in vitro grown Mycobacterium tuberculosis to lysozyme at concentrations between 0.1 and 3 mg/ml.2 Also, the acid fastness of in vivo bacteria varies, in a manner dependent upon their growth state (2). Considered together, these phenomena point to the need to understand the cell wall physical spatial organization and how the cell wall changes during in vivo growth.Mycobacterium leprae cannot be cultured in vitro and is propagated in nine-banded armadillos (Dasypus novemcinctus) (3). In this animal, as in the mouse foot pad model and human lepromatous leprosy, the bacteria grow logarithmically but with a very slow generation time of 12-14 days (4). Although there is a robust humoral response to M. leprae in armadillo, it would appear that the immune system does little to slow the growth of the bacteria and that the slow growth rate is an intrinsic property of the highly attenuated M. leprae, marked by less than 50% genomic coding capacity (5). This is in contrast to M. tuberculosis that presents an initial doubling time in animal models of about 2.4 days (5) u...

Centaurea maculosa Lam. is a noxious weed in western North America that produces a phytotoxin, (+/-)-catechin, which is thought to contribute to its invasiveness. Areas invaded by C. maculosa often result in monocultures of the weed, however; in some areas, North American natives stand their ground against C. maculosa and show varying degrees of resistance to its phytotoxin. Two of these resistant native species, Lupinus sericeus Pursh and Gaillardia grandiflora Van Houtte, were found to secrete increased amounts of oxalate in response to catechin exposure. Mechanistically, we found that oxalate works exogenously by blocking generation of reactive oxygen species in susceptible plants and reducing oxidative damage generated in response to catechin. Furthermore, field experiments show that L. sericeus indirectly facilitates native grasses in grasslands invaded by C. maculosa, and this facilitation can be correlated with the presence of oxalate in soil. Addition of exogenous oxalate to native grasses and Arabidopsis thaliana (L.) Heynh grown in vitro alleviated the phytotoxic effects of catechin, supporting the field experiments and suggesting that root-secreted oxalate may also act as a chemical facilitator for plant species that do not secrete the compound.