Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Mol Microbiol. 2008 Sep 18;70(3):762–774. doi: 10.1111/j.1365-2958.2008.06445.x

Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: Biosynthesis and Impact on the Persistence in mice

Tounkang Sambou 1,2,*, Premkumar Dinadayala 1,2,*, Gustavo Stadthagen 3,+, Nathalie Barilone 3, Yann Bordat 3,++, Patricia Constant 1,2, Florence Levillain 1,2, Olivier Neyrolles 1,2, Brigitte Gicquel 3, Anne Lemassu 1,2, Mamadou Daffé 1,2,‡, Mary Jackson 3,†,‡
PMCID: PMC2581643  NIHMSID: NIHMS72918  PMID: 18808383

SUMMARY

Mycobacterium tuberculosis and other pathogenic mycobacterial species produce large amounts of a glycogen-like α-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, orthologs 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 α-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 α-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.

Keywords: mycobacteria, tuberculosis, glucan, glycogen, polysaccharides, virulence

INTRODUCTION

Tuberculosis remains a serious health problem worldwide, with an estimated one third of the population infected by Mycobacterium tuberculosis, the causative agent of tuberculosis, and a death toll of more than 2 million people annually. Understanding the molecular mechanisms enabling the tubercle bacillus to multiply in a hostile environment such as the macrophage and to survive in the host is important for the design of novel strategies to combat the disease. The cell envelope of pathogenic mycobacteria plays important roles in host-pathogen interactions and in the resistance of these microorganisms to chemotherapeutic treatments (Daffé and Draper, 1998; Jarlier and Nikaido, 1990). The envelope consists of a typical plasma membrane, surrounded by a complex wall of carbohydrates and lipids that are organized into an outer membrane (Hoffmann et al., 2008; Zuber et al., 2008), which is in turn surrounded by layer (Daffé and Draper, 1998). The unusual chemical nature of the envelope makes it difficult for the host to degrade.

The outermost compartment of the cell envelope of pathogenic mycobacterial species consists in a loosely-bound structure called capsule (Chapman et al. 1959; Daffé and Draper, 1998; Hanks, 1961). We have previously shown that the capsular material is composed of carbohydrates and proteins with only small amounts of species- or type-specific lipids (Lemassu et al. 1996, Ortalo-Magné et al. 1995). Although mycobacteria shed some of this material in the culture medium during in vitro growth (Ortalo-Magné et al. 1995), capsular components clearly coat in vivo-grown bacteria (Schwebach et al., 2002), probably retained by the phagosomal membrane (Daffé and Draper, 1998). The major carbohydrate constituent of the capsule of M. tuberculosis, representing up to 80% of the extracellular polysaccharides, is a high molecular weight (>100,000 Da) α-glucan composed of a → 4- α-D-Glc-1 → core branched at position 6 every 5 or 6 residues by → 4- α-D-Glc-1 → oligoglucosides (Dinadayala et al., 2004; Lemassu and Daffé, 1994, Ortalo-Magné et al., 1995). Because the carbohydrate nature of the main constituent of the surface of pathogenic mycobacterial species has been determined much later than the discovery of the mycobacterial capsule (Dinadayala et al., 2004; Lemassu and Daffé, 1994, Lemassu et. al., 1996; Ortalo-Magné et al., 1995), there is at present little information about the biological functions of these components. Using Complement Receptor 3 (CR3)-transfected Chinese hamster ovary cells, Cywes and collaborators showed that capsular polysaccharides, among which the α-glucan, mediated the non-opsonic binding of M. tuberculosis H37Rv to CR3 (Cywes et al., 1997). Given that CR3 is one of the principal phagocytic receptors of monocytes and neutrophils and that CR3-mediated phagocytosis can result in the diminution or absence of oxidative burst and suppression of IL-12 secretion, it was proposed that this route of entry may be favourable to the intracellular survival of the tubercle bacillus (Ehlers and Daffé, 1998; Fenton et al., 2005). In another study, capsular components of M. tuberculosis were shown to contain compounds that displayed anti-phagocytic properties with certain types of macrophages (Stokes et al., 2004). α-glucan was also recently shown to induce monocytes to differentiate into altered dendritic cells that failed to present lipid antigens to CD1-restricted T cells, to upregulate CD80, and that produced IL10 but not IL-12 (Gagliardi et al. 2007). Finally, the cell wall α-glucan of Pseudallescheria boydii, which appears to be similar in structure to that of pathogenic mycobacteria, was involved in the internalization of this fungus by macrophages and the induction of the innate immune response by a mechanism dependent on toll-like receptor 2, CD14 and MyD88 (Bittencourt et al., 2006).

Although these findings suggested an important role for capsular glucan in the regulation of phagocytosis and the modulation of the immune response, most of them were derived from in vitro studies using purified glucan and cellular models that may not accurately reflect its contribution during mycobacterial infections. The construction of mutants deficient in the synthesis of glucan was thus warranted to assess the relevance and individual contribution of this polysaccharide to the pathogenicity of M. tuberculosis.

RESULTS

Identification of M. tuberculosis H37Rv genes potentially involved in α-D-glucan and glycogen biosynthesis

M. tuberculosis and M. bovis BCG capsular glucans are composed of repeating units of → 4-α-D-Glcp-1 → residues and some → 4- α-D-Glcp substituted at position 6 with one to six α-D-Glcp residues (Lemassu and Daffé, 1994, Ortalo-Magné et al., 1995; Dinadayala et al., 2004), indicating a glycogen-like highly branched structure. Given the structural similarities between α-D-glucan and glycogen, the synthesis of both polysaccharides is expected to involve similar catalytic reactions (Fig. 1). The two polysaccharides may even share, at least in part, a common biosynthetic pathway. In addition to the α-1,4 glucosyltransferase gene Rv3032 we recently showed to be involved in the synthesis of both 6-O-methylglucosyl lipopolysaccharides (MGLP) and glycogen (Stadthagen et al., 2007), four genes were found to potentially participate in the synthesis of α-D-glucan and/or glycogen in the genome of M. tuberculosis H37Rv. Rv1213 encodes a putative ADPGlc pyrophosphorylase. The product of this gene shares 35% amino-acid identity with the E. coli ADPGlc pyrophosphorylase (GlgC) that is responsible for the synthesis of the glucosyl donor, ADP-Glc. Rv1213, whose ortholog in Corynebacterium glutamicum was recently implicated in glycogen synthesis (Tzvetkov et al., 2003), is immediately adjacent to the putative glycogen synthase gene, glgA (Rv1212c) that encodes an 1->4 glycosyl transferase. Rv1326c encodes an α-1,4-glucan branching enzyme potentially responsible for the branching of the oligoglucosyl units on the glucosyl backbone (Garg et al., 2007). Rv1326c shares 48% amino-acid identity (63% similarity) with the branching enzyme from E. coli. This M. tuberculosis glgB-like gene is arranged in an operon with glgE (Rv1327c), a gene encoding a glucanase whose ortholog in M. smegmatis is required for the degradation and recycling of glycogen (Belanger and Hatfull, 1999). It is also adjacent to another catabolic gene, glgP (Rv1328), encoding a probable glycogen phosphorylase (Schneider et al., 2000). Finally, Rv1562c (treZ) is the third gene of an operon that has been involved in the metabolism of trehalose (De Smet et al., 2000; Woodruff et al., 2004). The sequence similarity Rv1562c shares with bacterial α-1,4-glucan branching enzymes (26% identity with the E. coli GlgB protein on a 241 amino acid overlap) suggested that it may also catalyze the branching of glycogen and/or α-D-glucan.

Figure 1.

Figure 1

Open in a new tab

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

Disruption of the glgA, glgB, glgC and treZ ortholog genes by allelic exchange in M. tuberculosis H37Rv

glgA, glgC and treZ orthologs were disrupted by homologous recombination in M. tuberculosis H37Rv using standard protocols (Pelicic et al., 1997, Jackson et al., 2001). Allelic replacement at the glgA, glgC and treZ loci was confirmed in each mutant by Southern hybridization (Fig. S1). One glgA mutant, one treZ mutant and one glgC mutant were selected for further studies and named H37RvΔglgA, H37RvΔtreZ and H37RvΔglgC, respectively. Unexpectedly, despite repeated attempts to knock-out the glgB gene in M. tuberculosis H37Rv, no candidate allelic exchange mutants could be isolated. Single crossover was, however, achievable at the glgB locus (data not shown). To determine whether the failure to disrupt the glgB gene was due to its essentiality, we then performed an allelic exchange experiment using a M. tuberculosis merodiploid strain. This strain was constructed by transforming the clone that had undergone a single crossover at the glgB locus with pVVglgBtb. Gene replacement was then carried out following standard procedures (Jackson et al., 2001). Analysis by PCR and Southern blotting of eight clones corresponding to putative double crossover recombinants showed that allelic exchange had occurred at the chromosomal glgB locus of five of them (Fig. S1). Therefore, the expression of glgB from pVVglgBtb was sufficient to rescue an M. tuberculosis knock-out mutant, indicating that this gene is essential for growth. Allelic replacement at the glgB locus of M. tuberculosis was also achievable in the presence of a wild-type copy of the glgB gene from E. coli expressed from pVVglgBcoli, suggesting that both glgB orthologs have a similar function. Finally, in spite of individual glgA and Rv3032 knock-outs being achievable (Stadthagen et al., 2007), two independent attempts to disrupt the Rv3032 gene in H37RvΔglgA in order to create a mutant simultaneously deficient in both α-1,4 glucosyltransferases turned out to be unsuccessful. Thus, M. tuberculosis H37Rv requires a functional copy of at least one of these two genes for growth.

Structural features of the glycogen of M. tuberculosis

The structural features of the α-glucans from M. tuberculosis and M. bovis were previously established (Dinadayala et al., 2004; Lemassu and Daffé, 1994). The polysaccharide from both origins exhibited a glycogen-like structure, i.e. α → 4- α-D-Glcp-1 → core, substituted at some 6 positions with short α → 4- α-D-Glcp-1 → oligoglucoside chains. As these similarities were based on the structures of glycogens from non-mycobacterial sources, we first investigated the structure of M. tuberculosis glycogen. Knowing that the structural features of glycogen are age-dependent (Antoine & Tepper, 1969a), the mycobacterial α-glucan and intracellular glycogen analyzed herein were purified from the same culture batches. The α-glucan was extracted from culture filtrates as described (Lemassu and Daffé, 1994). The corresponding cells were disrupted and the intracellular glycogen was recovered from the cytosolic fraction as described under Experimental Procedures. The mycobacterial glycogen run in a Bio-Gel P-200 gel gave a single chromatographic peak at a position corresponding to an apparent molecular mass of 100,000 Da, as reported for the α-glucan of M. tuberculosis (Lemassu and Daffé, 1994; Ortalo-Magné et al., 1995). Surprisingly, this molecular mass was hundred-fold less than that estimated earlier by analytical ultracentrifugation (Antoine and Tepper, 1969b).

Gas chromatography (GC) analysis of the trimethysilyl derivatives of the acid hydrolysis products of the purified glycogen showed that it was exclusively composed of Glc. GC-mass spectrometry (GC-MS) analysis of the partially O-methylated, partially O-acetylated alditols derived from the polysaccharide showed that it consisted of the three expected types of residues, i.e. (i) terminal Glc-(1→; (ii) →4)-Glcp-(1 → or 5-linked-Glcf; and (iii) →4,6)-Glcp-(1 → or 5,6-Glcf. The pyranosyl conformation of the glycosyl residues that composed the polysaccharide was established from the examination of the 13C-NMR spectra (Fig. S2) that showed a signal at 100.5 ppm corresponding to the resonances of anomeric carbons in α-glucopyranosyl units (Dinadayala et al., 2004, Zang et al., 1991). The 1H-NMR spectrum of the M. tuberculosis glycogen (Fig. S2) was similar to that published for the α-glucan of M. bovis BCG (Dinadayala et al., 2004) and eukaryotic glycogen (Zang et al., 1991).

The lengths of the 6-branched chains in the glycogen from M. tuberculosis were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS of per-O-acetylated fragments released after digestion with pullulanase, an enzyme that specifically hydrolyzes Glc-α(1→6)-Glc linkages (Dinadayala et al., 2004). The efficiency of the enzymatic hydrolysis was monitored by the disappearance of the signal resonances at 5.00 ppm in the 1H-NMR spectra. The reaction was found to be complete after 72 hrs of digestion. The digestion of pullulan into tri-glucosides was used as the positive control whereas maltoheptaose, from which no degradation was observed, was the negative control. The digestion products of the glycogen from M. tuberculosis consisted mainly of two to six residues; minor oligosaccharides with seven to nine units were also present in the mixtures of per-O-acetylated oligosaccharides analyzed (Fig. S3). No oligosaccharide derivatives containing more ten or more sugar residues were detected. These data indicated that the lengths of the branched chains in the glycogen of M. tuberculosis are similar to those of the glucan from the same species (Lemassu and Daffé, 1994).

1H-NMR signals at 5.38 ppm, which correspond to the resonances of terminal, branched or linear α-D-Glcp-(1→4) anomeric protons, as well as those at 5.00 ppm assignable to the resonances of 4-linked or terminal α-D-Glcp-(1→6) anomeric protons, were observed in the spectra of glycogen (Fig. S2) and α-glucan (Dinadayala et al., 2004). The optical rotatory ([α]D) values measured for M. tuberculosis glycogen was 165° ± 5, and thus significantly different from that determined for M. bovis BCG glycogen (75°), indicating a difference in the conformation of the two polysaccharides (Dinadayala et al., 2008). Consistent with this, the dynamic radius of the glycogen of M. tuberculosis (25−26 nm) was different from that measured for the glycogen from M. bovis (35 nm, Dinadayala et al., 2008).

Glycogen and α-D-glucan contents of the M. tuberculosis knock-out mutants

To analyze the impact of the disruption of the various genes on the production of glucan, two complementary approaches were used. The relative percentages of Ara, Glc and Man, the sugar constituents of the extracellular arabinomannan glucan and mannan (Lemassu and Daffé, 1994) were determined by GC in the culture filtrates of three to four independent batches of the wild-type and mutant strains. Additionally, the amounts estimated by GC analysis of the three sugar constituents of the extracellular polysaccharides were calculated relative to an external standard, erythritol, (Ery). Very similar Glc contents (0.56 ± 0.07), determined as Glc versus Ara+Man+Glc ratios, were obtained for the different batches of the wild-type H37Rv strain (Fig. 2), indicating that the synthesis of capsular glucan was relatively constant and the preparation method reliable. Importantly, the ratios Glc/Ery varied according to the strains examined whereas those of Ara/Ery and Man/Ery were unchanged. Notably, two-fold less glucan was recovered from the culture medium of H37RvΔglgC (Fig. 2). The molecular radius of the glucan synthesized by H37RvΔglgC, which reflects the conformation of the polysaccharide, was also slightly (22 versus 26 nm) but significantly affected by the mutation (Table 1). Complementation of the mutant with a wild-type copy of glgC restored glucan production to wild-type level as well as the native structure of the polysaccharide (Fig. 2; Table 1). The production of glucan was similarly affected in the H37RvΔglgA mutant but not in H37RvΔRv3032 and H37RvΔtreZ (Fig. 2). Interestingly, glucan production could be restored in H37RvΔglgA upon complementation with a wild-type copy of the Rv3032 gene, indicating a partial redundancy of the two enzymes (Fig. 2). However, unlike the H37RvΔRv3032 mutant (Stadthagen et al., 2007), H37RvΔglgA produced wild-type amounts of MGLP (data not shown).

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

Figure 2

Open in a new tab

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)

Table 1.

Optical rotatory values ([α]D in degrees) and molecular radius (nm) of the glucans and glycogens purified from the wild-type, isogenic ΔglgC, ΔglgA and ΔRv3032 mutants, and complemented mutant strains of M. tuberculosis H37Rv. nd, not determined; nr, not relevant.

strain Glucan Glycogen
[α] D Radius (nm) [α] D Radius (nm)
H37Rv 212 ± 2 25.7 ± 1.1 167 ± 6 26.6 ± 0.6
H37RvΔglgC 186 ± 1 22.7 ± 0.7 134. ± 6 24.0 ± 1.0
H37RvΔglgC/pVVglgC 209 ± 8 27.9 ± 2.5 158. ± 6 27.5 ± 1.0
H37RvΔglgA 206 ± 5 23.8 ± 0.8 161 ± 15 25.6 ± 0.2
H37RvΔglgA/pVVglgA 208 ± 2 25.3 ± 0.5 173 ± 22 25.1 ± 1.1
H37RvΔglgA/pVVRv3032 204 ± 2 25.9 ± 1.4 nd nd
H37RvΔRv3032 215 ± 2 25.8 ± 0.7 166 ± 1 25.0 ± 0.1
H37RvΔRv3032/pVVRv3032 nr nr 166 ± 1 26.2 ± 1.1
H37RvΔtreZ nr 27.3 ± 0.7 nd 24.2 ± 0.6
Open in a new tab

Determination of the glycogen content of the wild-type and mutant strains indicated that both the H37RvΔglgC and H37RvΔRv3032 strains were affected in the production of this polysaccharide. Complementation of the mutants with the wild-type copy of the corresponding disrupted gene restored, either fully or partially, glycogen production (Fig. 3). The global structural features of the glycogens produced by the mutants were otherwise similar to those of M. tuberculosis H37Rv as determined by 1H-NMR analyses (data not shown) and dynamic light scattering (Table 1). Finally, no significant changes were observed in the glycogen content and structure of the H37RvΔglgA (Fig. 3) and H37RvΔtreZ mutants (data not shown).

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

Figure 3

Open in a new tab

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).

Altogether, our results thus indicated that (i) the ADP-Glc pyrophosphorylase GlgC is involved in the synthesis of both glucan and glycogen in M. tuberculosis although the GlgC-dependent ADPGlc pathway is not the only route to the production of glycogen/glucan in M. tuberculosis; (ii) GlgA and Rv3032 have partially redundant α-1,4-glucosyltransferase activities although GlgA is preferentially utilized to synthesize glucan whereas Rv3032 is predominantly used in the synthesis of glycogen and MGLP (Stadthagen et al., 2007); and (iii) TreZ is involved in the biosynthesis of trehalose (De Smet et al., 2000; Woodruff et al., 2004) but not in that of glucan and glycogen.

Glycogen and α-D-glucan contents of the merodiploid M. tuberculosis glgB knock-out mutants

GlgB appears to be an essential enzyme of M. tuberculosis. Allelic replacement at the glgB locus was, however, achievable in the presence of wild-type copies of the glgB genes from M. tuberculosis or E. coli expressed from the multi-copy plasmid pVV16 (see above). With the goal of establishing a link between GlgB and glucan and/or glycogen production, we thus set out to determine whether the deregulated expression of the glgB genes in the merodiploid strains and the expression of a heterologous glgB gene in H37RvΔglgB/pVVglgBcoli had any impact on the production and structural features of glucan and glycogen. Interestingly, the production of both the extracellular glucan and the intracellular glycogen was affected in H37RvΔglgB/pVVglgBtb. This strain synthesized only 57% and 36% of the glucan and glycogen normally produced by H37Rv, respectively (Table 2). Moreover, while these polysaccharides were eluted from the permeation gel at a similar position as those isolated from the parental strain, contained only Glc and displayed NMR spectra superimposable to those of wild-type glucan and glycogen, their conformations were significantly different from those of wild-type H37Rv. The molecular radiuses measured for the glycogen and the glucan of H37RvΔglgB/pVVglgBtb were significantly lower than those of the native polysaccharides (Table 2). Furthermore, the rotatory power values of both polysaccharides were affected in the merodiploid strain (Table 2). Although the merodiploid H37RvΔglgB/pVVglgBcoli produced amounts of glycogen and glucan comparable to those of the wild-type strain, the conformation of glycogen was also significantly affected by the mutation (Table 2). Results thus indicated that, in vivo, the GlgB enzyme from M. tuberculosis (i) is involved in the biosynthesis of both glucan and glycogen and (ii) has the same function as its E. coli counterpart.

Table 2.

Quantification and conformational analyses of glycogens and glucans from the wild-type and merodiploid ΔglgB mutants of M. tuberculosis H37Rv. nd, not determined.

Strain Glucan Glycogen
Glc/(Glc+Ara+Man) [α]d Radius(nm) amount/wt [α]d Radius (nm)
H37Rv 0.56 ± 0.07 211 ± 2 25.7 ± 1.1 100 ± 2 167 ± 6 26.6 ± 0.6
H37RVΔglgB/pVVglgBtb 0.32 ± 0.02 127 ± 3 21.5 ± 0.4 36 ± 8 44 ± 1 22.5 ± 0.1
H37RVΔglgB/pVVglgBcoli 0.54 ± 0.05 nd 27.3 ± 0.5 97 ± 9 91 ± 3 30.0 ± 1.6
Open in a new tab

Multiplication and persistence of the glg mutants in cultured BALB/c mouse bone-marrow macrophages and in mice organs

To analyze the effects of disrupting glg genes on the virulence of M. tuberculosis H37Rv we used both BALB/c bone-marrow macrophages and mouse infection models. We first evaluated the possible influence of the α-glucan content on the uptake and intracellular growth of the H37RvΔglgA mutant and its parental strains in cultured mouse macrophages. Indeed, the α-glucan has been implicated in the CR3-mediated phagocytosis of M. tuberculosis, a route of entry that may be favourable to the intracellular survival of the tubercle bacillus (Ehlers and Daffé, 1998; Fenton et al., 2005). However, the H37RvΔglgA mutant, which produces two-fold less glucan than did the wild-type strain, did not showed any defects in the examined parameters in both resting and activated mouse cells (Fig. S4). As α-glucan was also recently shown to impact on monocyte differentiation into dendritic cells, which then produce IL10 but not IL-12 and fail to present lipid antigens to CD1-restricted T cells and to upregulate CD80 (Gagliardi et al. 2007) it was important to determine the putative impact of the disruption of glg genes on the virulence of M. tuberculosis H37Rv in the context of the whole animal. Accordingly, the ability of the wild-type and H37RvΔglA, H37RvΔtreZ, H37RvΔglgC and H37RvΔRv3032 mutant strains to replicate and persist in the lungs and spleen of BALB/c mice was studied by CFU analysis. As shown in Fig. 4, bacterial organ burdens in the mice infected with H37RvΔtreZ, H37RvΔglgC and H37RvΔRv3032 were comparable to those of mice infected with the parental H37Rv strain throughout a 90- to 100-day period. In contrast, the ability of H37RvΔglgA to persist in both organs during the late stage of infection was significantly reduced. At 42 and 90 days post-infection, the lungs of mice infected with the H37RvΔglgA mutant carried about than 10-fold less CFU than those of mice infected with the control M. tuberculosis H37Rv strain.

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

Figure 4

Open in a new tab

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.

DISCUSSION

The first purpose of the present study was to identify the genes involved in the biosynthesis of the capsular glucan and intracellular glycogen of M. tuberculosis. We reasoned, based on the structures of the two polysaccharides, that their biosynthetic pathways was likely to be very similar to that established for bacterial glycogen in general. Accordingly, putative orthologs of the glgA, glgB and glgC genes of E. coli and Bacillus subtilis were identified in the genome of M. tuberculosis H37Rv. Inactivation of glgC (Rv1213) resulted in an important reduction in the production of both glucan and glycogen, whereas disruption of the putative α-1,4-glucosyltransferase genes glgA (Rv1212c) and Rv3032 specifically affected glucan and glycogen synthesis, respectively. Given the very similar structures of glycogen and glucan, the molecular mechanisms underlying the preferred involvement of GlgA and Rv3032 in one or the other pathway is unknown. A possible mechanism may be related to the putative participation of the two enzymes in different macromolecular complexes, one being dedicated to the cytosolic biosynthesis of glycogen, the other – perhaps coupled to an export system - to that of glucan. Clearly, further studies will be required to verify this hypothesis. Our data indicate, however, that Rv3032 and GlgA can at least partially compensate for one another and that no other α-1,4-glucosyltransferase participates in the biosynthesis of glycogen and glucan in M. tuberculosis. This conclusion is based on the three observations that (i) both H37RvΔglgA and H37RvΔRv3032 still produced glycogen and glucan; (ii) the overexpression of Rv3032 in H37RvΔglgA fully restored glucan synthesis; and (iii) at least one of the two α-1,4-glucosyltransferase genes needs to be functional for M. tuberculosis to sustain growth in vitro. A correlate of these results is that the production of glycogen, glucan and/or MGLP, is an essential physiological requirement of the tubercle bacillus. In this context, it is interesting to note that while not all mycobacterial species display functional glgA and Rv3032 genes, they all have retained at least one of the two enzymes. M. leprae, for instance, which produces MGLP (Hunter et al., 1986), carries an ortholog of Rv3032 in its genome, but glgA is a pseudogene. Conversely, a frameshift mutation at the 5’ end of the Rv3032 gene of M. smegmatis causes this gene to be inactive (our unpublished results). M. smegmatis does, however, express a glgA gene that most likely accounts for the production of MGLP in this species.

The fact that significant amounts of both glucan and glycogen were still produced in the ADPGlc pyrophosphorylase glgC mutant indicates that, unlike the situation in E. coli (Preiss and Romeo, 1989), the GlgC-dependent ADPGlc pathway is not the only route to glycogen/glucan synthesis in M. tuberculosis H37Rv. Other as yet unidentified enzymes with low sequence similarity to the usual prokaryotic ADPGlc pyrophosphorylases may exist in M. tuberculosis that compensate, at least partially, for the loss of GlgC in the mutant. Alternatively, one of the two α-1,4-glucosyltransferases, Rv3032 or GlgA, might utilize UDPGlc instead of ADPGlc as the sugar donor in the elongation reactions. Providing support for this assumption, our preliminary studies on the substrate specificity of Rv3032 indicate that this enzyme has the ability to transfer Glc from UDPGlc onto short-chain glucooligosaccharides in vitro (G. Stadthagen, M. Jackson, unpublished results). Finally, the presence of a putative amylomaltase gene (Rv1781c, malQ) in the M. tuberculosis genome suggests that this bacterium might have the ability to synthesize α-1,4-glucans when grown on maltose or maltodextrin (Preiss and Romeo, 1989).

An important result of this study is the demonstrated essentiality of the branching enzyme GlgB. This result was unexpected as GlgB from M. tuberculosis is, to our knowledge, the first glycogen-branching enzyme reported to be essential in a prokaryotic organism. The successful complementation of H37RvΔglgB with the glgB gene of E. coli proves that the GlgB proteins from both organisms have, in vivo, similar catalytic functions. This result is consistent with the recently demonstrated branching activity of GlgB from M. tuberculosis in an in vitro assay (Garg et al., 2007). The reason(s) of the essentiality of this enzyme in the tubercle bacillus is (are) unknown. As GlgB seems to be the only branching enzyme committed in the synthesis of glucan and glycogen, its essentiality may be related to the physiological requirement of M. tuberculosis to produce at least one of these two polysaccharides as suggested above. By comparison with glgB mutants of E. coli (Lares et al., 1974), one may also speculate that a deficiency in GlgB could lead to the accumulation of linear poorly water-soluble polymers of 4-linked α-glucosyl residues either in the cytosol or in the periplasmic space of the cell envelope (in the case of a putative precursor of the capsular α-glucan being exported) causing the death of the Mycobacterium. The latter mechanism would be similar to the one thought to account for the lethal character of the counterselectable marker sacB in Gram negative bacteria, mycobacteria and Corynebacteria, when sucrose is present in the culture medium (Reyrat et al., 1998). The levansucrase SacB catalyzes the formation of polymers of fructose from sucrose, which accumulation in the periplasm is believed to lead to cell death.

The Rv3032 and treZ mutants were not affected in their multiplication or persistence in the BALB/c mouse model. The virulent phenotype of the latter mutant is consistent with the virulence data reported by Murphy and collaborators on a treY mutant of M. tuberculosis H37Rv affected in the same trehalose pathway (Murphy et al. 2005). In contrast, the glgA mutant was significantly affected in its ability to persist in both the spleen and the lungs of mice. In view of the biochemical phenotype of this mutant and of the existence of two GlgA-independent pathways for the synthesis of trehalose in mycobacteria (de Smet et al., 2000; Murphy et al., 2005), we postulate that the virulence attenuation of the glgA mutant is due to a decrease in the production of capsular glucan, not in that of glycogen or trehalose. Two hypotheses may then account for the absence of an attenuation phenotype associated to H37RvΔglgC which displays in vitro a similar reduction in glucan content as H37RvΔglgA. Firstly, an increase in UDPGlc production and UDPGlc-dependent activity of Rv3032 may compensate for the absence of (or decrease in) ADPGlc in the bacterial cell during host infection. Secondly, α-1,4 glucan synthesis from maltose or maltodextrin may be stimulated during the persistence stage of infection. Efforts are now ongoing in our laboratories to generate an M. tuberculosis mutant specifically deficient in the synthesis of the capsular glucan through the targeted inactivation of the transport system(s) of this extracellular polysaccharide.

EXPERIMENTAL PROCEDURES

Bacterial strains and growth conditions

E. coli XL1-blue, the strain used for cloning experiments, was propagated in Luria Bertani (LB) broth (pH 7.5) (Becton Dickinson, Sparks, MD). M. tuberculosis H37Rv was grown at 37°C in Middlebrook 7H9 broth supplemented with 10% ADC enrichment (Difco) and 0.05% Tween 80, in minimal Sauton's medium as surface pellicles or on agar Middlebrook 7H11 medium supplemented with OADC (Difco). When required, antibiotics were added at the following concentrations: kanamycin, 20 μg/ml; hygromycin B, 50 μg/ml. For selection of allelic exchange mutants, 2% sucrose was added to the solid medium.

Construction of the treZ, glgB, glgA, glgC and glgA-Rv3032 mutants

The ts/sacB methodology (Pelicic et al., 1997) was used to achieve allelic replacement at the treZ (Rv1562c), glgC (Rv1213) and Rv3032 loci of M. tuberculosis H37Rv. The glgA (Rv1212c) gene was disrupted using a two-step homologous recombination procedure (Jackson et al., 2001). The essentiality of glgB (Rv1326c) was investigated following a standard strategy based on the construction of a merodiploid strain (Jackson et al., 2001). Standard PCR strategies were used to amplify the M. tuberculosis H37Rv treZ, glgB, glgA, Rv3032 and glgC genes. glgA and its flanking regions were amplified using primers glgA.1 (5’-gcggaattccgcggtcgcattttcacgtgg-3’) and glgA.2 (5’-gcggaattcggatggtcgacgaccatatcc-3’), and a disrupted allele of the glgA gene was constructed by cloning the kanamycin (Km) resistance cassette from pUC4K (Amersham Pharmacia Biotech) into the NcoI sites of glgA. Digestion by NcoI resulted in the deletion of 253 bp of the coding sequence of glgA. glgA::Km was then cloned into pJQ200-xylE, yielding pJQglgAKX, the plasmid used for allelic replacement. The glgB gene and flanking regions was amplified using primers B130.2 (5’-gcatttggtcctgaagaagctacg-3’) and B130.4 (5’-ggggatatccacagcgccgaagtgggcgg-3’), and a disrupted allele of the glgB gene was constructed by cloning the Km cassette into the SalI sites of glgB. SalI-cut resulted in the deletion of 740 bp of the coding sequence of glgB. glgB::Km was then cloned with the xylE colored marker (Pelicic et al., 1997) into the BamHI site of pJQ200 to yield pJQglgBKX, the construct used for allelic replacement at the glgB locus. The glgC gene and flanking regions was amplified using primers C364.1 (5’-cccgaattcggctgggatagacccgcaac-3’) and C364.2 (5’-cccgaattcggcgtttcatcagcagttcg-3’) and inserted into pGEM®-T easy (Promega), yielding pGEMTglgC. A disrupted allele of the glgC gene was then constructed by cloning the Km cassette into the BamHI site of glgC. glgC::Km was finally cloned with the xylE gene into the BamHI site of pPR27 (Pelicic et al., 1997), to yield p27glgCKX. treZ was amplified using the couple of primers B48.2 (5’-gcttcctgggcggcgcataccatc-3’) and B48.3 (5’-cccgaattcggccggctccgcagcccgcag-3’) and a disrupted allele of the treZ gene was constructed by cloning the Km cassette into the XhoI site of treZ. treZ::Km was then cloned with the xylE gene into the BamHI site of pPR27, yielding p27treZKX. The Rv3032 gene and flanking regions were PCR amplified from M. tuberculosis H37Rv genomic DNA using primers Rv3032.1 (5’-gggctgcagatcgccggcgcgctggcc-3’) and Rv3032.2 (5’-tgagccatgtcgcctccctgg-3’) and a disrupted allele, Rv3032::Str, was obtained by inserting a streptomycin resistance cassette into the SmaI restriction site of Rv3032. Rv3032::Str was then cloned into the NotI-cut and blunt-ended pPR27-xylE to obtain pPR27Rv3032SX, the construct used for allelic replacement in H37RvΔglgA.

Complementation of the glgA, glgB, glgC and treZ mutants

Wild-type copies of the M. tuberculosis H37Rv glgA, glgB, glgC and treZ genes carried by the mycobacterial expression plasmid pVV16 (Jackson et al., 2000), in pVVglgA, pVVglgBtb, pVVglgC and pVVtreZ, respectively, were used for complementation studies. The primers used to amplify glgC were glgC.1 (5'-gggctgcagccatgagagaagtgccgcac-3') and glgC.2 (5'-cccaagcttgatccaaacacccttgcccacg-3'). glgZ.2 (5’-tggtgggcatatgcctgaattccgagtatgggc3’) and glgZ.3 (5'-gggaagcttggccggctccgcagccc gcag-3') were used to amplify treZ. glgA.5 (5’-ccgcgcgcatatgcgggtggcgatgttgactc-3’) and glgA.6 (5’-cccaagcttcgcgcacaccttccggtagatg-3’) were used to amplify glgA. glgB.1 (5’-cccccccccatatgagtcgatccgagaaactcacc-3’) and glgB.2 (5’-gggaagcttggcgggcgtcagccacagcgc-3’) were used to amplify glgB. In addition, the glgB gene from E. coli was PCR-amplified using the primers glgBc.5 (5’-ggccgggcatatgtccgatcgtatcgatagagac-3’) and glgBc.6 (5’-cccaagcttttctgcctcccgaaccagccag-3’) and cloned into pVV16, yielding pVVglgBcoli. The primers were designed to generate PCR products corresponding to the entire genes devoid of their stop codon and harboring NdeI or PstI and HindIII restriction sites (underlined in the primers’ sequences), enabling direct cloning into pVV16. In this vector, genes are constitutively expressed under control of the hsp60 transcription and translation signals and the recombinant proteins produced carry a six-histidine tag at their carboxyl terminus. The production of recombinant proteins in the complemented mutants was analyzed by immunoblotting with the monoclonal Penta-His antibody from Qiagen as described previously (Jackson et al., 2000).

Analysis of glycogen and glucan

For the analysis of glycogen and α-D-glucan, M. tuberculosis strains were grown in Sauton's medium as surface pellicles. High molecular weight carbohydrates and proteins were precipitated from ten fold-concentrated culture filtrates with six volumes of cold ethanol overnight at 4°C. Precipitates were then recovered by centrifugation at 14,000 × g for one hr, dissolved in distilled water, dialyzed for one day against water to eliminate traces of salts and glycerol, lyophilized and weighed. The extracellular α-D-glucan content was determined by GC of sugar derivatives resulting from the acid hydrolysis of the lyophilized macromolecules. To this end, 100 μg of polysaccharides and 20 μg of erythritol (used as an internal standard) were hydrolyzed in 200 μl of 2 M trifluoroacetic acid for 1 hr at 110°C and then dried under nitrogen. The resulting free hydroxyl groups of liberated sugars were trimethylsilylated as described (Sweeley et al., 1963), solubilized in petroleum ether and analyzed by GC. Further purification of α-D-glucan was carried out by chromatography on a (7 × 1 cm) DEAE-trisacryl gel (IBF, Villeneuve-La-Garenne, France). The neutral fractions, eluted with 0.01 M NH4Cl (pH = 8.3), were re-chromatographed on a Bio-Gel P-60 column (Bio-Rad) (90 × 1.8 cm) using 0.5% acetic acid as the eluent and refractive index detection. The purity of the excluded fractions was checked by 1H-NMR analysis as described previously (Dinadayala et al., 2004).

For the purification of glycogen, heat-killed cells were weighed, suspended in water and centrifuged twice at 1,100 × g for 15 min to remove loosely attached compounds among which the extracellular α-D-glucan. Washed cells were then broken in a French press at 140 bars to liberate the intracellular glycogen. The material was centrifuged at 1,100 × g for 30 min to eliminate non-broken cells and the supernatant was re-centrifuged at 27,000 × g for 15 min to remove cell-walls. Glycogen was extracted as described by Bueding and Orrell (1964) with only minor modifications. Briefly, one volume of supernatant was mixed with three volumes of 0.2 M glycine buffer (pH 10.5) and two volumes of chloroform at 4°C. The upper phase was removed and the lower phase was re-extracted three times with two volumes of glycine buffer. The aqueous phases were pooled, concentrated, and centrifuged at 100,000 × g for 4 hrs at 4°C. The gelatinous pellet was homogenized in 15 ml of water, shaken for 15 min with 5 ml of chloroform/1-octanol (3:1, by vol.), and decanted. The upper glycogen phase was recovered and the treatment with chloroform/1-octanol was repeated for longer periods, i.e. 30 and 150 min, until the proteinaceous interphase disappeared. The aqueous phase was then precipitated with six volumes of cold ethanol overnight at 4°C, centrifuged for 1 hr at 1,500 × g, lyophilized and weighted. The purification process was monitored by determining the carbohydrate content of the fractions and by 1H-NMR analysis. The glycogen content was defined by the mass of glycogen versus mass of wet cells.

Samples of polysaccharides purified from the wild-type and mutant strains were comparatively O-methylated (Blakeney and Stone, 1985); portions of the per-O-methylated products were hydrolyzed with 2 M CF3COOH at 110 °C for 2 h, reduced with NaBH4 and acetylated. The different partially O-methylated and partially O-acetylated glucitols were identified by GC and GC-MS.

Samples of polysaccharides purified from the wild-type and mutant strains were comparatively hydrolyzed with pullulanase and the resulting fragments were O-acetylated and analyzed by MALDI-TOF mass spectrometry as described (Dinadayala et al., 2004, 2008)

MALDI-TOF mass spectra (in the positive mode) were acquired on a Voyager-DE STR mass spectrometer (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser emitting at 337 nm. Samples were analyzed in the Reflector mode using an extraction delay time set at 100 ns and an accelerating voltage operating in positive ion mode of 20 kV. To improve the signal-to-noise ratio, 150 single shots were averaged for each mass spectrum and typically, four individual spectra were accumulated to generate a summed spectrum. Mass spectrum calibration was performed using the calibration mixture 1 of Sequazyme™ Peptide Mass Standards Kit (Perseptive Biosystems), including known peptide standards in a mass range from 900 Da to 1600 Da. Internal mass calibration was performed as previously described (Laval et al. 2001). Stock solutions of peracetylated oligosides were prepared in chloroform, at a concentration of 1mM and were directly applied on the sample plate as 1 μl droplets, followed by the addition of 0.5 μl matrix solution [2,5-dihydroxybenzoic acid (10 mg/ml) in CHCl3/CH3OH (1 :1, v/v)]. Samples were allowed to crystallize at room temperature.

The 1H- and 13C-NMR analyses were performed on a Bruker AMX-500 spectrometer at 500.13 MHz and 125.77 MHz, respectively, using a 5mm BBI probe, at 343°K in D2O. A basic pulse program was used for 1H-NMR spectra, without presaturation of HOD signal, in order to preserve the integration of the signal at 5.00 ppm. A pulse of 45° was applied before a 2 s recycle delay. FID was recorded on 16k points, and processed by a sine-bell apodization function before Fourier transformation and integration. Bruker DEPT (distortionless enhancement by polarization transfer) sequence was applied for 13C-NMR analyses.

GC of monosaccharide derivatives was performed on a Hewlett-Packard 5890 series II apparatus equipped with an OV1 capillary column (0.30mm × 25m) using helium gas. The temperature separation program involved an increase from 100° to 300°C at the rate of 5°C/min, followed by 10 min at 300°C.

GC-MS analyses were performed on a HP 5889X mass spectrometer (electron energy, 70 eV) coupled to a HP 5890 series II gas chromatograph fitted with a column identical to that used for GC. GC-MS analyses were performed in the electron impact mode.

Conformational studies by dynamic light scattering

The diffusion parameters of the purified polysaccharide solutions (1 mg/ml in distilled water) were determined by DLS. The apparatus consisted of a DynaPro-MS/X Dynamic light scattering instrument (ProteinSolutions, Charlottesville, VA), providing an 830 nm light beam. Samples were analyzed in a 12 μl quartz sample cell at 20°C. Using the Dynamic V6 software, DLS autocorrelation function were collected at a scattering angle of 90° with a data acquisition time of 200s.

M. tuberculosis growth and persistence in mice

Female BALB/c mice (6- to 8-week-old) purchased from CERJanvier (Le Genest St Isle, France) were infected through the aerosol route with approximately 100 CFUs of wild-type M. tuberculosis H37Rv or the treZ and glgC mutant strains. M. tuberculosis aerosols were generated from bacterial suspensions consisting of 5 × 106 CFUs per ml in phosphate-buffered saline. Mice were exposed to the aerosols for 15 min. Mice were infected intravenously with 105 CFU of wild-type M. tuberculosis or the glgA and Rv3032 mutants as described (Jackson et al., 1999). Five mice were used per experimental point and per strain.

Infection of BALB/c bone marrow derived macrophages with M. tuberculosis

BALB/c bone marrow macrophages were prepared, activated or not, and infected at a multiplicity of infection of one bacillus per macrophage as described (Rousseau et al., 2004).

Statistics

Statistical significance was determined using paired Student's t-tests.

Acknowledgements

We thank Dr Françoise Laval for her help in mass spectrometry. This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Institut Pasteur (GPH 05-Tuberculose), the National Institute of Allergy and Infectious Diseases/ National Institutes of Health grant R01AI064798, the European Commission contract no. LSHP-CT-2005-018923 (NM4TB) and the CONACyT, Mexico (fellowship no. 175523 to G. S.). The NMR equipment was funded via European structural funds (FEDER), the CNRS (France), the University Paul Sabatier (Toulouse) and the “Région Midi-Pyrénées” funds as part of the 2000−2006 CPER program.

REFERENCES

  1. Antoine AD, Tepper BS. Environmental control of glycogen and lipid content of Mycobacterium tuberculosis. J. Bacteriol. 1969a;100:538–539. doi: 10.1128/jb.100.1.538-539.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Antoine AD, Tepper BS. Characterization of glycogens from mycobacteria. Arch. Biochem. Biophys. 1969b;134:207–213. doi: 10.1016/0003-9861(69)90267-7. [DOI] [PubMed] [Google Scholar]
  3. Belanger AE, Hatfull GF. Exponential-phase glycogen recycling is essential for growth of Mycobacterium smegmatis. J. Bacteriol. 1999;181:6670–6678. doi: 10.1128/jb.181.21.6670-6678.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blakeney AB, Stone BA. Methylation of carbohydrates with lithium methylsulphinyl carbanion. Carbohydrate Res. 1985;140:319–324. [Google Scholar]
  5. Bittencourt VCB, Figueiredo RT, da Silva RB, Mourao-Sa DS, Fernandez PL, Sassaki GL, Mulloy B, Bozza MT, Barreto-Bergter E. An α-glucan of Pseudallesscheria boydii is involved in fungal phagocytosis and Toll-like receptor activation. J. Biol. Chem. 2006;281:22614–22623. doi: 10.1074/jbc.M511417200. [DOI] [PubMed] [Google Scholar]
  6. Bueding E, Orrell S. A mild procedure for the isolation of polydisperse glycogen from animal tissues. J. Biol. Chem. 1964;239:4018–4020. [PubMed] [Google Scholar]
  7. Chapman GB, Hanks JH, Wallace JH. An electron microscope study of the disposition and fine structure of Mycobacterium lepraemurium in mouse spleen. J. Bacteriol. 1959;77:205–211. doi: 10.1128/jb.77.2.205-211.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cywes C, Hoppe HC, Daffé M, Ehlers MRW. Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent. Infect. Immun. 1997;65:4258–4266. doi: 10.1128/iai.65.10.4258-4266.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Daffé M, Draper P. The envelope layers of mycobacteria with reference to their pathogenicity. Advances in microbial physiology. 1998;39:131–203. doi: 10.1016/s0065-2911(08)60016-8. [DOI] [PubMed] [Google Scholar]
  10. De Smet KAL, Weston A, Brown IN, Young DB, Robertson BD. Three pathways for trehalose biosynthesis in mycobacteria. Microbiology. 2000;146:199–208. doi: 10.1099/00221287-146-1-199. [DOI] [PubMed] [Google Scholar]
  11. Dinadayala P, Lemassu A, Granovski P, Cérantola S, Winter N, Daffé M. Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis bacille Calmette-Guérin. J. Biol. Chem. 2004;279:12369–12378. doi: 10.1074/jbc.M308908200. [DOI] [PubMed] [Google Scholar]
  12. Dinadayala P, Sambou T, Daffé M, Lemassu A. Comparative structural analyses of the α-glucan and glycogen from Mycobacterium bovis. Glycobiology. 2008 doi: 10.1093/glycob/cwn031. in press. [DOI] [PubMed] [Google Scholar]
  13. Ehlers MRW, Daffé M. Interactions between Mycobacterium tuberculosis and host cells: are mycobacterial sugars the key? Trends in Microbiology. 1998;6:328–335. doi: 10.1016/s0966-842x(98)01301-8. [DOI] [PubMed] [Google Scholar]
  14. Fenton MJ, Riley LW, Schlesinger LS. Receptor-mediated recognition of Mycobacterium tuberculosis by host cells. In: Cole ST, Davis Eisenach K, McMurray DN, Jacobs WR Jr., editors. Tuberculosis and the tubercle bacillus. ASM Press; Washington, DC: 2005. pp. 405–426. [Google Scholar]
  15. Gagliardi MC, Lemassu A, Teloni R, Mariotti S, Sargentini V, Pardini M, Daffe M, Nisini R. Cell wall-associated alpha-glucan is instrumental for Mycobacterium tuberculosis to block CD1 molecule expression and disable the fucntion of dendritic cells derived from infected monocyte. Cell Microbiol. 2007;9:2081–2092. doi: 10.1111/j.1462-5822.2007.00940.x. [DOI] [PubMed] [Google Scholar]
  16. Garg SK, Alam MS, Kishan KVR, Agrawal P. Expression and characterization of α-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv. Protein Expr. Purif. 2007;51:198–208. doi: 10.1016/j.pep.2006.08.005. [DOI] [PubMed] [Google Scholar]
  17. Hanks JH. The origin of the capsules on Mycobacterium leprae and other tissue-grown mycobcteria. Int. J. Leprosy. 1961;26:172–174. [Google Scholar]
  18. Hunter SW, Gaylord H, Brennan PJ. Structure and antigenicity of the phosphorylated lipopolysaccharide antigens from the leprosy and tubercle bacilli. J. Biol. Chem. 1986;261:12345–12351. [PubMed] [Google Scholar]
  19. Hoffmann C, Leis A, Niederweis M, Plitzko JM, Engelhardt H. Disclosure of the mycobacterial outer membrane: cryo-electron tomography and vitreous sections reveal the lipid bilayer structure. Proc. Natl. Acad. Sci. U S A. 2008;105:3963–3967. doi: 10.1073/pnas.0709530105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jackson M, Phalen SW, Lagranderie M, Ensergueix D, Chavarot P, Marchal G, McMurray DN, Gicquel B, Guilhot C. Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine. Infect. Immun. 1999;67:2867–2873. doi: 10.1128/iai.67.6.2867-2873.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jackson M, Crick DC, Brennan PJ. Phosphatidylinositol is an essential phospholipid of mycobacteria. J. Biol. Chem. 2000;275:30092–30099. doi: 10.1074/jbc.M004658200. [DOI] [PubMed] [Google Scholar]
  22. Jackson M, Camacho LR, Gicquel B, Guilhot C. Gene replacement and transposon delivery using the negative selection marker sacB. In: Parish T, Stocker NG, editors. Mycobacterium tuberculosis protocols. Vol. 54. Humana Press; Totowa N J: 2001. pp. 59–75. [DOI] [PubMed] [Google Scholar]
  23. Lares C, Frixon C, Creuzet-Sigal N. Characterization and ultrastructure of mutants of Escherichia coli deficient in α-1,4-glucan- α-1,4-glucan 6-glycosyltransferase (branching enzyme). J. Gen. Microbiol. 1974;82:279–293. doi: 10.1099/00221287-82-2-279. [DOI] [PubMed] [Google Scholar]
  24. Laval F, Lanéelle MA, Déon C, Monsarrat B, Daffé M. Accurate Molecular Mass Determination of Mycolic acids by MALDI-TOF Mass Spectrometry. Anal. Chem. 2001;73:4537–4544. doi: 10.1021/ac0105181. [DOI] [PubMed] [Google Scholar]
  25. Lemassu A, Daffé M. Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis. Biochem. J. 1994;297:351–357. doi: 10.1042/bj2970351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lemassu A, Ortalo-Magné A, Bardou F, Silve G, Lanéelle MA, Daffé M. Extracellular and surface-exposed polysaccharides of non-tuberculous mycobacteria. Microbiology. 1996;142:1513–1520. doi: 10.1099/13500872-142-6-1513. [DOI] [PubMed] [Google Scholar]
  27. Murphy HN, Stewart GR, Mischenko VV, Apt AS, Harris R, McAlister MSB, Driscoll PC, Young DB, Robertson BD. The OtsAB pathway is essential for trehalose biosynthesis in Mycobacterium tuberculosis. J. Biol. Chem. 2005;280:14524–14529. doi: 10.1074/jbc.M414232200. [DOI] [PubMed] [Google Scholar]
  28. Jarlier V, Nikaido H. Permeability barrier to hydrophilic solutes in Mycobacterium chelonei. J. Bacteriol. 1990;172:1418–1423. doi: 10.1128/jb.172.3.1418-1423.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ortalo-Magné A, Dupont MA, Lemassu A, Andersen AB, Gounon P, Daffé M. Molecular composition of the outermost capsular material of the tubercle bacillus. Microbiol. 1995;141:1609–1620. doi: 10.1099/13500872-141-7-1609. [DOI] [PubMed] [Google Scholar]
  30. Pelicic V, Jackson M, Reyrat JM, Jacobs WR, Jr, Gicquel B, Guilhot C. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA. 1997;94:10955–10960. doi: 10.1073/pnas.94.20.10955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Preiss J, Romeo T. Physiology, biochemistry and genetics of bacterial glycogen synthesis. Adv. Microb. Physiol. 1989;30:183–238. doi: 10.1016/s0065-2911(08)60113-7. [DOI] [PubMed] [Google Scholar]
  32. Reyrat J-M, Pelicic V, Gicquel B, Rappuoli R. Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect. Immun. 1998;66:4011–4017. doi: 10.1128/iai.66.9.4011-4017.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rousseau C, Winter N, Pivert E, Bordat Y, Neyrolles O, Avé P, Huerre M, Gicquel B, Jackson M. Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection. Cell. Microbiol. 2004;6:277–287. doi: 10.1046/j.1462-5822.2004.00368.x. [DOI] [PubMed] [Google Scholar]
  34. Schneider D, Bruton CJ, Chater KF. Duplicated gene clusters suggest an interplay of glycogen and trehalose metabolism during sequential stages of aerial mycelium development in Streptomyces coelicolor A3(2). Mol. Gen. Genet. 2000;263:543–553. doi: 10.1007/s004380051200. [DOI] [PubMed] [Google Scholar]
  35. Schwebach JR, Glatman-Freedman A, Gunther-Cummins L, Dai Z, Robbins JB, Schneerson R, Casadevall A. Glucan is a component of the Mycobacterium tuberculosis surface that is expressed in vitro and in vivo. Infect. Immun. 2002;70:2566–2575. doi: 10.1128/IAI.70.5.2566-2575.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stadthagen G, Sambou T, Guerin M, Barilone N, Boudou F, Kordulakova J, Charles P, Alzari PM, Lemassu A, Daffé M, Puzo G, Gicquel B, Rivière M, Jackson M. Genetic basis for the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis. J. Biol. Chem. 2007;282:27270–27276. doi: 10.1074/jbc.M702676200. [DOI] [PubMed] [Google Scholar]
  37. Stokes RW, Norris-Jones R, Brooks DE, Beveridge TJ, Doxsee D, Thorson LM. The glycan-rich outer layer of the cell wall of Mycobacterium tuberculosis acts as an antiphagocytic capsule limiting the association of the bacterium with macrophages. Infect. Immun. 2004;72:5676–5686. doi: 10.1128/IAI.72.10.5676-5686.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sweeley CC, Bentley R, Makita M, Wells WW. Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. J. Am. Chem. Soc. 1963;85:2497–2507. [Google Scholar]
  39. Tzvetkov M, Klopprogge C, Zelder O, Liebl W. Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and altered cell wall lipid composition. Microbiology. 2003;149:1659–1673. doi: 10.1099/mic.0.26205-0. [DOI] [PubMed] [Google Scholar]
  40. Woodruff PJ, Carlson BL, Siridechadilok B, Pratt MR, Senaratne RH, Mougous JD, Riley LW, Williams SJ, Bertozzi CR. Trehalose is required for growth of Mycobacterium smegmatis. J. Biol. Chem. 2004;279:28835–28843. doi: 10.1074/jbc.M313103200. [DOI] [PubMed] [Google Scholar]
  41. Zang LH, Howseman AM, Shulman RG. Assignment of the 1H chemical shifts of glycogen. Carbohydrate Res. 1991;220:1–9. doi: 10.1016/0008-6215(91)80001-4. [DOI] [PubMed] [Google Scholar]
  42. Zuber B, Chami M, Houssin C, Dubochet J, Griffiths G, Daffé M. Direct visualization of the outer membrane of native mycobacteria and corynebacteria. J. Bacteriol. 2008 doi: 10.1128/JB.01919-07. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES