Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope

Abstract

SummaryThe antigen 85 complex of Mycobacterium tuberculosis consists of three abundantly secreted proteins. The recent characterization of a mycoloyltransferase activity associated in vitro with each of these antigens suggested that they are potentially important for the building of the unusual cell envelope of mycobacteria. To define the physiological role of these proteins, the gene coding for antigen 85C was inactivated by transposon mutagenesis. The resulting mutant was shown to transfer 40% fewer mycolates to the cell wall with no change in the types of mycolates esterifying arabinogalactan or in the composition of non-covalently linked mycolates. As a consequence, the diffusion of the hydrophobic chenodeoxycholate and the hydrophilic glycerol, but not that of isoniazid, was found to be much faster through the cell envelope of the mutant than that of the parent strain. Taken together, these data demonstrate that: (i) antigen 85C is involved directly or indirectly in the transfer of mycolates onto the cell wall of the whole bacterium; (ii) the enzyme is not specific for a given type of mycolate; and (iii) the cell wall-linked mycolate layer may represent a barrier for the diffusion of small hydrophobic and hydrophilic molecules.

The impact of the absence of glycopeptidolipids on the ultrastructure, cell surface and cell wall properties, and phagocytosis of Mycobacterium smegmatis

“…These data indicated that the diffusion of these antibiotics through the cell wall barrier was not the limiting step for their activity. A similar observation has been made for the fbpC-inactivated mutant of Mycobacterium tuberculosis, which exhibited a change in its cell wall permeability to chenodeoxycholate due to a 40 % reduction of covalently linked mycolates (Jackson et al, 1999).…”

“…Although no sign of a second lipid bilayer has ever been reported in thin sections of mycobacterial cells (Draper, 1998), freeze-fractured samples of mycobacteria (Barksdale & Kim, 1977 ;Benedetti et al, 1984 ;Takeo et al, 1984) showed that these organisms had two such planes of weakness in their envelopes ; in addition to the expected plasma membrane fracture, a second fracture plane, close to the cell surface of mycobacteria, was observed. The cell wall-linked mycolates certainly participate in this barrier since the disruption of a gene that encodes a mycoloyltransferase, namely antigen-85C, causes a decrease in the amount of cell wall-bound mycolates and affects the permeability of the envelope of the mutant (Jackson et al, 1999). Evidence has also been presented that the chemical structure of mycolic acids plays a role in determining the fluidity and permeability of the mycobacterial cell wall (George et al, 1995 ;Liu et al, 1996 ;Dubnau et al, 2000).…”

“…The permeability of the strains of M. smegmatis to chenodeoxycholate was assessed as previously described (Bardou et al, 1998 ;Jackson et al, 1999). Exponentially grown cells (2-day-old cultures) were first labelled for 16 h with [5,6-$H]uracil (20 µM, 1n85 GBq mmol − " ; DuPont NEN) to quantify the biomass present in aliquots used in the accumulation assays.…”

A Role of thecspAGene Encoding a Mycolyltransferase in the Growth under Alkaline Conditions ofCorynebacterium glutamicum

“…It has been reported that the Mycobacterium tuberculosis mutant of the fbpA gene, a homolog of C. glutamicum cspA, showed increased uptake of hydrophilic glycerol and of hydrophobic chenodeoxycholate. 29) It has also been reported that the M. tuberculosis mutant of the kasB gene involved in mycolic acid synthesis showed increased sensitivity to several antibiotics. The C. glutamicum cspA mutant strain also showed a faster uptake rate of hydrophilic compounds, including glycerol and acetate.…”

“…33) It has also been reported that the M. tuberculosis fbpA mutant showed faster uptake of hydrophobic chenodeoxycholate. 29) These phenomena are to be explained by analogy with the outer membrane of Gram-negative bacteria. Lipopolysaccharides occupy the outer leaflet of the outer membrane and form a highly ordered quasicrystaline structure with very low fluidity.…”