Molecular composition of the outermost capsular material of the tubercle bacillus

Abstract

To gain insight into the pathogenesis of tuberculosis, a molecular definition of the tubercle bacillus cell envelope, which is involved in the early stages of the infection, is required. The cell-surface-exposed material of the pathogen was isolated by mechanical means and chemically analysed. It was shown by scanning electron microscopy that the method used for extracting the surfacecovering material preserves the integrity of the bacilli. Surprisingly, in view of the current opinion, only small amounts of lipids ( 1 4 % ) were present. Polysaccharides and proteins were the main components of the material. The polysaccharides were neutral and lipid-free D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 120,13 and 4 kDa, respectively. Based on NMR spectroscopy and conventional chemical analyses, the major structural motifs of the purified polysaccharides were established as being identical to those of the polysaccharides we previously isolated from the culture filtrate of the tubercle bacillus. lmmunocytochemical studies showed that these compounds were not only surface-located but were also present in the inner capsular compartment. The major protein constituents exhibited the same mobilities on SDS-PAGE as those of the culture filtrate of the tubercle bacillus and readily reacted with the monoclonal antibodies directed against these molecules. These proteins included the 19 and 38 kDa lipoproteins, the 30/31 kDa fibronectin-binding proteins and the 40 kDa L-alanine dehydrogenase. These findings suggest that the culture filtrate material represents part of the capsule which, in an in wivo context, could contribute to the electron transparent zone surrounding the tubercle bacillus. The 24 kDa (MPBTT64) protein was found to be a secreted protein, as it was detected almost exclusively in the culture filtrate. Taken together, the data give a new insight into the surface-exposed compounds of the tubercle bacillus and may explain part of the nature and limitation of the host immunity towards the pathogen.

Surface-exposed Glycopeptidolipids of Mycobacterium smegmatis Specifically Inhibit the Phagocytosis of Mycobacteria by Human Macrophages

“…20). Some of these biologically active molecules have been precisely localized in the outermost layer of the cell envelope (15,21), but the precise location of others is still a matter of debate.…”

“…We extracted mycobacterial surface-exposed compounds by gentle surface abrasion with glass beads (15) and tested their role in the interactions between mycobacteria and MDMs to identify the most active bacterial partners of the phagocytic process. Because we have demonstrated that pathogenic and nonpathogenic strains use the same phagocytic receptors to infect cells (9,10,22,23), this work was performed with the nonpathogenic species, M. smegmatis.…”

Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase

“…In mycobacteria, a polysaccharide-protein capsule [13] surrounds the peptidoglycan/arabinogalactan/mycolic acid cell wall [30]. Mycolic acids and free lipids are thought to contribute to the unusually low permeability of the mycobacterial cell wall which is believed to play a role in the resistance of mycobacteria to therapeutic agents.…”

“…The smooth glass beads treatment, releasing mainly the capsular material [13], contained between 5 and 10% of the total enzyme activity found in the soluble extract prepared after complete disruption of the cells. The steel beads preparation, containing components more deeply embedded in the cell envelope, presented about 20% of the total enzyme activity.…”

“…Isolation of the outermost cell-envelope material was developed by Ortalo-Magne  et al [13]. Briefly, 7 g of wet cells (14 days culture) were harvested by removing the medium and then gently shaking for 1 min with 35 g of glass beads (4 mm diameter).…”