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

Abstract

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...

Differential Detergent Extraction of Mycobacterium marinum Cell Envelope Proteins Identifies an Extensively Modified Threonine-Rich Outer Membrane Protein with Channel Activity

“…Although the existence of a second membrane was already suggested by Minnikin (2), it was only recently unequivocally shown as a lipid bilayer structure by cryoelectron tomography (3)(4)(5). As the observed thickness of the MOM is 7 to 8 nm, mycolic acids are proposed to be present in a folded conformation (4,6), which is in agreement with recent Langmuir assays (7). The outer leaflet of this membrane is probably formed by an array of unusual mycolate-or mycobacteriumspecific (glyco)lipids, such as trehalose dimycolate, phtiocerol dimycoserosate, and sulfolipids.…”

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

“…Mycobacterium leprae) vs. grown in vitro (i.e. M.tb) showing that bacilli grown in vivo had a more compact cell envelope with more mycolic acids and more but shorter arabinogalactan molecules per peptidoglycan (Bhamidi et al, 2011). This differential cell envelope spatial conformation may differentially impact the rearrangement of the outer surface exposed cell envelope components that have a critical role in M.tb-host recognition.…”

“…There are many models of the mycobacterial cell envelope (Bhamidi et al, 2011;Brennan and Besra, 1997;Crick et al, 2003;Dmitriev et al, 2000;Domenech et al, 2001). Interactions of the asymmetric plasma membrane, peptidoglycan, and covalent attached arabinogalactan together with LAM and PIMs have been speculated, at least some of which are known to be associated with the plasma membrane.…”

GtrA Protein Rv3789 Is Required for Arabinosylation of Arabinogalactan in Mycobacterium tuberculosis

“…Alditol acetates were prepared from mAGP or AGP, as described previously (26), with minor modifications. Briefly, 10 g of heptitol or scyllo-inositol, serving as the internal standards, was added to 0.1 to 1 mg of dry mAGP or AGP.…”