Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice

Abstract

Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like alpha-glucan that represents the major polysaccharide of their outermost capsular layer. To determine the role of the surface-exposed glucan in the physiology and virulence of these bacteria, orthologues of the glg genes involved in the biosynthesis of glycogen in Escherichia coli were identified in M. tuberculosis H37Rv and inactivated by allelic replacement. Biochemical analyses of the mutants and complemented strains indicated that the synthesis of glucan and glycogen involves the alpha-1,4-glucosyltransferases Rv3032 and GlgA (Rv1212c), the ADP-glucose pyrophosphorylase GlgC (Rv1213) and the branching enzyme GlgB (Rv1326c). Disruption of glgC reduced by half the glucan and glycogen contents of M. tuberculosis, whereas the inactivation of glgA and Rv3032 affected the production of capsular glucan and glycogen, respectively. Attempts to disrupt Rv3032 in the glgA mutant were unsuccessful, suggesting that a functional copy of at least one of the two alpha-1,4-glucosyltransferases is required for growth. Importantly, the glgA mutant was impaired in its ability to persist in mice, suggesting a role for the capsular glucan in the persistence phase of infection. Unexpectedly, GlgB was found to be an essential enzyme.

Figure 1

Proposed pathway for the biosynthesis of glycogen and glucan in M. tuberculosis.

Figure 2. Glucan contents of M. tuberculosis H37Rv, its isogenic H37RvΔglgA, H37RvΔglgC, H37RvΔRv3032 and H37RvΔtreZ mutants, and the complemented mutant strains

Exocellular polysaccharides were isolated from 3 to 4 different batches, hydrolyzed by trifluoroacetic acid, trimethylsililated and analyzed by GC, using erythritol as an internal standard. The percentages of glucose in the monosaccharide mixture were determined. The values correspond to the mean of at least 3 different batches. Asterisks (**) denote P values < 0.01 (Student's t-test)

Figure 3. Glycogen contents of M. tuberculosis H37Rv, its isogenic H37RvΔglgA, H37RvΔglgC and H37RvΔRv3032 mutants, and the complemented mutant strains

The intracellular glycogens were isolated from disrupted cells originated 3 to 4 independent batches, purified as described under Experimental Procedures and weighed. The quantity of glycogen per g of bacterial dry mass was determined and compared to the value in the parental H37Rv strain arbitrary set to 100 %. The values correspond to the mean of at least 3 different experiments. Asterisks (**) denote P values < 0.01 (Student's t-test).

Figure 4. Multiplication and persistence of wild-type H37Rv, H37RvΔtreZ, H37RvΔglgC, H37RvΔRv3032 and H37RvΔglgA in the lungs and spleen of BALB/c mice

Results are expressed as means ± standard deviations (error bars) of CFU counts for five infected mice. Mice were infected either intravenously (H37Rv, H37RvΔRv3032, H37RvΔglgA) or via the aerosol route (H37Rv, H37RvΔtreZ, H37RvΔglgC). Asterisks denote P values < 0.05 (Student's t-test) for a comparison of H37Rv versus H37RvΔglgA.