LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain.

BACKGROUND: Aminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region. RESULTS: Using genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human ß-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria. CONCLUSIONS: Overall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.

Bioinformatic Mining and Structure-Activity Profiling of Baeyer-Villiger Monooxygenases from Mycobacterium tuberculosis.

Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), one of the deadliest infectious diseases. The alarming health context coupled with the emergence of resistant M. tuberculosis strains highlights the urgent need to expand the range of anti-TB antibiotics. A subset of anti-TB drugs in use are prodrugs that require bioactivation by a class of M. tuberculosis enzymes called Baeyer-Villiger monooxygenases (BVMOs), which remain understudied. To examine the prevalence and the molecular function of BVMOs in mycobacteria, we applied a comprehensive bioinformatic analysis that identified six BVMOs in M. tuberculosis, including Rv3083 (MymA), Rv3854c (EthA), Rv0565c, and Rv0892, which were selected for further characterization. Homology modeling and substrate docking analysis, performed on this subset, suggested that Rv0892 is closer to the cyclohexanone BVMO, while Rv0565c and EthA are structurally and functionally similar to MymA, which is by far the most prominent type I BVMO enzyme. Thanks to an unprecedented purification and assay optimization, biochemical studies confirmed that all four BVMOs display BV-oxygenation activity. We also showed that MymA displays a distinctive substrate preference that we further investigated by kinetic parameter determination and that correlates with in silico modeling. We provide insights into distribution of BVMOs and the structural basis of their substrate profiling, and we discuss their possible redundancy in M. tuberculosis, raising questions about their versatility in prodrug activation and their role in physiology and infection. IMPORTANCE Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of death worldwide. The rise in drug resistance highlights the urgent need for innovation in anti-TB drug development. Many anti-TB drugs require bioactivation by Baeyer-Villiger monooxygenases (BVMOs). Despite their emerging importance, BVMO structural and functional features remain enigmatic. We applied a comprehensive bioinformatic analysis and confirmed the presence of six BVMOs in M. tuberculosis, including MymA, EthA, and Rv0565c-activators of the second-line prodrug ethionamide-and the novel BVMO Rv0892. Combining in silico characterization with in vitro validation, we outlined their structural framework and substrate preference. Markedly, MymA displayed an enhanced capacity and a distinct selectivity profile toward ligands, in agreement with its catalytic site topology. These features ground the molecular basis for structure-function comprehension of the specificity in these enzymes and expand the repertoire of BVMOs with selective and/or overlapping activity for application in the context of improving anti-TB therapy.

Evidence for amylose inclusion complexes with multiple acyl chain lipids using solid-state NMR and theoretical approaches.

Amylose is known to form inclusion complexes in the presence of hydrophobic guests. Among lipids, only single-chain fatty acids have been reported as possible guests with the surrounding amylose in a well-defined V-helix conformation. Using experimental 13C solid-state NMR, we studied the formation of inclusion complexes between amylose and a variety of multiple-chains lipids of increasing complexity. Molecular dynamics simulations and calculations of 13C isotropic chemical shifts using the Density Functional Theory approach were performed to support the interpretation of experimental spectra. We provide unambiguous evidences that amylose forms inclusion complexes with lipids bearing multiple acyl chains. Amylose conformations around these lipids are characterized by {ϕ,ψ} anomeric bond dihedral angles near {115°,105°}. In the 13C NMR spectra, this translates into C1 and C4 chemical shifts of 102.5 ppm and 81.1 ppm, regardless of the helical conformation of the amylose surrounding the acyl chains.

Lipid and Lipoarabinomannan Isolation and Characterization.

The very high content of structurally diverse and biologically active lipids of exotic structures is the hallmark of Mycobacteria. As such the lipid composition is commonly used to characterize mycobacterial strains at the species and type-species levels. The present chapter describes the methods that allow the purification of the most commonly isolated biologically active lipids and those used for analyzing extractable lipids and their constituents, cell wall-linked mycolic acids (MA), and lipoarabinomannan (LAM). These involve various chromatographic techniques and analytical procedures necessary for structural and metabolic studies of mycobacterial lipids. In addition, as the use of physical methods has brought important overhang on chemical structures of the very-long-chain MA, which typify mycobacteria, NMR and mass spectrometry data of these specific fatty acids are included.

Discovery of a novel dehydratase of the fatty acid synthase type II critical for ketomycolic acid biosynthesis and virulence of Mycobacterium tuberculosis.

The fatty acid synthase type II (FAS-II) multienzyme system builds the main chain of mycolic acids (MAs), important lipid pathogenicity factors of Mycobacterium tuberculosis (Mtb). Due to their original structure, the identification of the (3 R)-hydroxyacyl-ACP dehydratases, HadAB and HadBC, of Mtb FAS-II complex required in-depth work. Here, we report the discovery of a third dehydratase protein, HadDMtb (Rv0504c), whose gene is non-essential and sits upstream of cmaA2 encoding a cyclopropane synthase dedicated to keto- and methoxy-MAs. HadDMtb deletion triggered a marked change in Mtb keto-MA content and size distribution, deeply impacting the production of full-size molecules. Furthermore, abnormal MAs, likely generated from 3-hydroxylated intermediates, accumulated. These data strongly suggest that HadDMtb catalyzes the 3-hydroxyacyl dehydratation step of late FAS-II elongation cycles during keto-MA biosynthesis. Phenotyping of Mtb hadD deletion mutant revealed the influence of HadDMtb on the planktonic growth, colony morphology and biofilm structuration, as well as on low temperature tolerance. Importantly, HadDMtb has a strong impact on Mtb virulence in the mouse model of infection. The effects of the lack of HadDMtb observed both in vitro and in vivo designate this protein as a bona fide target for the development of novel anti-TB intervention strategies.

HadD, a novel fatty acid synthase type II protein, is essential for alpha- and epoxy-mycolic acid biosynthesis and mycobacterial fitness.

Mycolic acids (MAs) have a strategic location within the mycobacterial envelope, deeply influencing its architecture and permeability, and play a determinant role in the pathogenicity of mycobacteria. The fatty acid synthase type II (FAS-II) multienzyme system is involved in their biosynthesis. A combination of pull-downs and proteomics analyses led to the discovery of a mycobacterial protein, HadD, displaying highly specific interactions with the dehydratase HadAB of FAS-II. In vitro activity assays and homology modeling showed that HadD is, like HadAB, a hot dog folded (R)-specific hydratase/dehydratase. A hadD knockout mutant of Mycobacterium smegmatis produced only the medium-size alpha'-MAs. Data strongly suggest that HadD is involved in building the third meromycolic segment during the late FAS-II elongation cycles, leading to the synthesis of the full-size alpha- and epoxy-MAs. The change in the envelope composition induced by hadD inactivation strongly altered the bacterial fitness and capacities to aggregate, assemble into colonies or biofilms and spread by sliding motility, and conferred a hypersensitivity to the firstline antimycobacterial drug rifampicin. This showed that the cell surface properties and the envelope integrity were greatly affected. With the alarmingly increasing case number of nontuberculous mycobacterial diseases, HadD appears as an attractive target for drug development.

Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria.

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl ß-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide.

Mycobacteria have a complex cell wall structure that includes many lipids; however, even within a single subspecies of Mycobacterium avium these lipids can differ. Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide (L5P), yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis as well as biochemical and physico-chemical approaches. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in ovine strains, compared to five modules in bovine strains (C-type). Sequence analysis predicted these modules would produce the tripeptide Phe-N-Methyl-Val-Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico-chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D-Phe-N-Methyl-L-Val-L-Ala-OMe attached in N-ter to a 20-carbon fatty acid chain. These data demonstrate that S-type strains, which are more adapted in sheep, produce a unique lipid. There is a dose-dependent effect observed for L3P on upregulation of CD25+ CD8 T cells from infected cows, while L5P effects were static. In contrast, L5P demonstrated a significantly stronger induction of CD25+ B cells from infected animals compared to L3P.

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two ß subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.

Trehalose Polyphleates Are Produced by a Glycolipid Biosynthetic Pathway Conserved across Phylogenetically Distant Mycobacteria.

Mycobacteria synthesize a variety of structurally related glycolipids with major biological functions. Common themes have emerged for the biosynthesis of these glycolipids, including several families of proteins. Genes encoding these proteins are usually clustered on bacterial chromosomal islets dedicated to the synthesis of one glycolipid family. Here, we investigated the function of a cluster of five genes widely distributed across non-tuberculous mycobacteria. Using defined mutant analysis and in-depth structural characterization of glycolipids from wild-type or mutant strains of Mycobacterium smegmatis and Mycobacterium abscessus, we established that they are involved in the formation of trehalose polyphleates (TPP), a family of compounds originally described in Mycobacterium phlei. Comparative genomics and lipid analysis of strains distributed along the mycobacterial phylogenetic tree revealed that TPP is synthesized by a large number of non-tuberculous mycobacteria. This work unravels a novel glycolipid biosynthetic pathway in mycobacteria and extends the spectrum of bacteria that produce TPP.

The changes in mycolic acid structures caused by hadC mutation have a dramatic effect on the virulence of Mycobacterium tuberculosis.

Understanding the molecular strategies used by Mycobacterium tuberculosis to invade and persist within the host is of paramount importance to tackle the tuberculosis pandemic. Comparative genomic surveys have revealed that hadC, encoding a subunit of the HadBC dehydratase, is mutated in the avirulent M. tuberculosis H37Ra strain. We show here that mutation or deletion of hadC affects the biosynthesis of oxygenated mycolic acids, substantially reducing their production level. Additionally, it causes the loss of atypical extra-long mycolic acids, demonstrating the involvement of HadBC in the late elongation steps of mycolic acid biosynthesis. These events have an impact on the morphotype, cording capacity and biofilm growth of the bacilli as well as on their sensitivity to agents such as rifampicin. Furthermore, deletion of hadC leads to a dramatic loss of virulence: an almost 4-log drop of the bacterial load in the lungs and spleens of infected immunodeficient mice. Both its unique function and importance for M. tuberculosis virulence make HadBC an attractive therapeutic target for tuberculosis drug development.

The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis.

Gram positive mycobacteria with a high GC content, such as the etiological agent of tuberculosis Mycobacterium tuberculosis, possess an outer membrane mainly composed of mycolic acids (MAs), the so-called mycomembrane, which is essential for the cell. About thirty genes are involved in the biosynthesis of MAs, which include the hadA, hadB and hadC genes that encode the dehydratases Fatty Acid Synthase type II (FAS-II) known to function as the heterodimers HadA-HadB and HadB-HadC. The present study shows that M. smegmatis cells remain viable in the absence of either HadA and HadC or both. Inactivation of HadC has a dramatic effect on the physiology and fitness of the mutant strains whereas that of HadA exacerbates the phenotype of a hadC deletion. The hadC mutants exhibit a novel MA profile, display a distinct colony morphology, are less aggregated, are impaired for sliding motility and biofilm development and are more resistant to detergent. Conversely, the hadC mutants are significantly more susceptible to low- and high-temperature and to selective toxic compounds, including several current anti-tubercular drugs.

Increased Vancomycin Susceptibility in Mycobacteria: a New Approach To Identify Synergistic Activity against Multidrug-Resistant Mycobacteria.

Mycobacterium tuberculosis is wrapped in complex waxes, impermeable to most antibiotics. Comparing Mycobacterium bovis BCG and M. tuberculosis mutants that lack phthiocerol dimycocerosates (PDIM) and/or phenolic glycolipids with wild-type strains, we observed that glycopeptides strongly inhibited PDIM-deprived mycobacteria. Vancomycin together with a drug targeting lipid synthesis inhibited multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates. Our study puts glycopeptides in the pipeline of potential antituberculosis (TB) agents and might provide a new antimycobacterial drug-screening strategy.

Functional characterisation of three o-methyltransferases involved in the biosynthesis of phenolglycolipids in Mycobacterium tuberculosis.

Phenolic glycolipids are produced by a very limited number of slow-growing mycobacterial species, most of which are pathogen for humans. In Mycobacterium tuberculosis, the etiologic agent of tuberculosis, these molecules play a role in the pathogenicity by modulating the host immune response during infection. The major variant of phenolic glycolipids produced by M. tuberculosis, named PGL-tb, consists of a large lipid core terminated by a glycosylated aromatic nucleus. The carbohydrate part is composed of three sugar residues, two rhamnosyl units and a terminal fucosyl residue, which is per-O-methylated, and seems to be important for pathogenicity. While most of the genes responsible for the synthesis of the lipid core domain and the saccharide appendage of PGL-tb have been characterized, the enzymes involved in the O-methylation of the fucosyl residue of PGL-tb remain unknown. In this study we report the identification and characterization of the methyltransferases required for the O-methylation of the terminal fucosyl residue of PGL-tb. These enzymes are encoded by genes Rv2954c, Rv2955c and Rv2956. Mutants of M. tuberculosis harboring deletion within these genes were constructed. Purification and analysis of the phenolglycolipids produced by these strains, using a combination of mass spectrometry and NMR spectroscopy, revealed that Rv2954c, Rv2955c and Rv2956 encode the methyltransferases that respectively catalysed the O-methylation of the hydroxyl groups located at positions 3, 4 and 2 of the terminal fucosyl residue of PGL-tb. Our data also suggest that methylation at these positions is a sequential process, starting with position 2, followed by positions 4 and 3.

Structural elucidation and genomic scrutiny of the C60-C100 mycolic acids of Segniliparus rotundus.

Mycolic acids, very long-chain α-alkyl, ß-hydroxylated fatty acids, occur in the members of the order Corynebacteriales where their chain lengths (C(26)-C(88)) and structural features (oxygen functions, cis or trans double bonds, cyclopropane rings and methyl branches) are genus- and species-specific. The molecular composition and structures of the mycolic acids of two species belonging to the genus Segniliparus were determined by a combination of modern analytical chemical techniques, which include MS and NMR. They consist of mono-ethylenic C(62-)C(64) (α'), di-ethylenic C(77)-C(79) (α) and extremely long-chain mycolic acids (α(+)) ranging from 92 to 98 carbon atoms and containing three unsaturations, cis and/or trans double bonds and/or cyclopropanes. The double bonds in each class of mycolic acids were positioned by oxidative cleavage and exhibit locations similar to those of α- and α'-mycolic acids of mycobacteria. For the ultralong chain α-mycolic acids, the three double bonds were located at equally spaced carbon intervals (C(13)-C(16)), with the methyl branches adjacent to the proximal and distal trans double bonds. Examination of the Segniliparus rotundus genome compared with those of other members of the Corynebacteriales indicated two obvious differences in genes encoding the elongation fatty acid (FAS-II) enzymes involved in the biosynthesis of mycolic acids: the organization of 3-ketoacyl-ACP synthases (KasA and KasB) and (3R)-hydroxyacyl-ACP dehydratases (HadAB/BC), on one hand, and the presence of two copies of the hadB gene encoding the catalytic domain of the latter enzyme type, on the other. This observation is discussed in light of the most recent data accumulated on the biosynthesis of this hallmark of Corynebacteriales.

A novel mycolic acid species defines two novel genera of the Actinobacteria, Hoyosella and Amycolicicoccus.

Corynebacterineae are characterized by the presence of long-chain lipids, notably mycolic acids (α-alkyl, ß-hydroxy fatty acids), the structures of which are genus-specific. Mycolic acids from two environmental strains, Amycolicicoccus subflavus and Hoyosella altamirensis, were isolated and their structures were established using a combination of mass spectrometry analysis, (1)H-NMR spectroscopy and chemical degradations. The C(2)-C(3) cleavage of these C(30)-C(36) acids led to the formation of two fragments: saturated C(9)-C(11) acids, and saturated and unsaturated C(20)-C(25) aldehydes. Surprisingly, the fatty acids at the origin of the two fragments making up these mycolic acids were present in only minute amounts in the fatty acid pool. Moreover, the double bond in the main C(24) aldehyde fragment was located at position ω16, whereas that found in the ethylenic fatty acids of the bacteria was at ω9. These data question the biosynthesis of these new mycolic acids in terms of the nature of the precursors, chain elongation and desaturation. Nevertheless, they are consistent with the occurrence of the key genes of mycolic acid biosynthesis, including those encoding proteins of the fatty acid synthase II system, identified in the genome of A. subflavus. Altogether, while the presence of mycolic acids and analysis of their 16S rDNA sequences would suggest that these strains belong to the Mycobacteriaceae family, the originality of their structures reinforces the recent description of the novel genera Amycolicicoccus and Hoyosella.

Identification of mycobacterial alpha-glucan as a novel ligand for DC-SIGN: involvement of mycobacterial capsular polysaccharides in host immune modulation.

Mycobacterium tuberculosis possesses a variety of immunomodulatory factors that influence the host immune response. When the bacillus encounters its target cell, the outermost components of its cell envelope are the first to interact. Mycobacteria, including M. tuberculosis, are surrounded by a loosely attached capsule that is mainly composed of proteins and polysaccharides. Although the chemical composition of the capsule is relatively well studied, its biological function is only poorly understood. The aim of this study was to further assess the functional role of the mycobacterial capsule by identifying host receptors that recognize its constituents. We focused on alpha-glucan, which is the dominant capsular polysaccharide. Here we demonstrate that M. tuberculosis alpha-glucan is a novel ligand for the C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). By using related glycogen structures, we show that recognition of alpha-glucans by DC-SIGN is a general feature and that the interaction is mediated by internal glucosyl residues. As for mannose-capped lipoarabinomannan, an abundant mycobacterial cell wall-associated glycolipid, binding of alpha-glucan to DC-SIGN stimulated the production of immunosuppressive IL-10 by LPS-activated monocyte-derived dendritic cells. By using specific inhibitors, we show that this IL-10 induction was DC-SIGN-dependent and also required acetylation of NF-kappaB. Finally, we demonstrate that purified M. tuberculosis alpha-glucan, in contrast to what has been reported for fungal alpha-glucan, was unable to activate TLR2.

Identification of the polyketide synthase involved in the biosynthesis of the surface-exposed lipooligosaccharides in mycobacteria.

Lipooligosaccharides (LOS) are highly antigenic glycolipids produced by a number of Mycobacterium species, which include "M. canettii," a member of the M. tuberculosis complex, and the opportunistic pathogens M. marinum and M. kansasii. The various LOS share a core composed of trehalose esterified by at least 1 mole of polymethyl-branched fatty acid (PMB-FA) and differ from one another by their oligosaccharide extensions. In this study, we identified a cluster of genes, MSMEG_4727 through MSMEG_4741, likely involved in the synthesis of LOS in M. smegmatis. Disruption of MSMEG_4727 (the ortholog of pks5 of M. tuberculosis), which encodes a putative polyketide synthase, resulted in the concomitant abrogation of the production of both PMB-FA and LOS in the mutant strain. Complementation of the mutant with the wild-type gene fully restored the phenotype. We also showed that, in contrast to the case for "M. canettii" and M. marinum, LOS are located in deeper compartments of the cell envelope of M. smegmatis. The availability of two mycobacterial strains differing only in LOS production should help in defining the biological role(s) of this important glycolipid.

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

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.

Comparative structural analyses of the alpha-glucan and glycogen from Mycobacterium bovis.

Pathogenic mycobacteria such as Mycobacterium tuberculosis, the causative agent of tuberculosis, are surrounded by a noncovalently bound capsule, whose major carbohydrate constituent is a glycogen-like alpha-glucan. In the present study we compared the structures of the extracellular polysaccharide to that of the ubiquitous intracellular glycogen. The alpha-glucan was isolated from the culture medium of Mycobacterium bovis Bacille Calmette Guérin, the vaccine strain, in which it is released whereas the intracellular glycogen was obtained after the disruption of cells. The two purified polysaccharides were eluted from permeation gel at a similar position but glycogen was less soluble and gave a more opalescent solution in water than alpha-glucan. Combination of gas chromatography-mass spectrometry analysis of partially O-methylated, partially O-acetylated alditols and NMR analysis confirmed that both polysaccharides were composed of -->4-alpha-D-Glcp-1--> core, substituted at some six positions with short chains. Degradation of polysaccharides with pullulanase, followed by mass spectrometry analysis of the resulting products, also showed that the two polysaccharides do not differ in terms of lengths of branching. Interestingly, application of analytical ultracentrifugation and dynamic light scattering to the mycobacterial alpha-glucan and glycogen and their enzymatic degradative products indicated that the alpha-glucan possessed a higher molecular mass and was more compact than the glycogen from the same species, allowing the formulation of working structural models for the two polysaccharides. Consistent with the models, the alpha-glucan was found to be less accessible to pullulanase, a debranching enzyme, than glycogen.