The EAL domain containing protein STM2215 (rtn) is needed during Salmonella infection and has cyclic di-GMP phosphodiesterase activity.

Salmonella Typhimurium gene STM2215 (rtn) is conserved among many enterobacteriaceae. Mutants lacking STM2215 poorly colonized the liver and spleen in intraperitoneal infection of mice and poorly colonized the intestine and deeper tissues in oral infection. These phenotypes were complemented by a wild-type copy of STM2215 provided in trans. STM2215 deletion mutants grew normally in J774A.1 murine macrophages but were unable to invade Caco-2 colonic epithelial cells. Consistent with this finding, mutants in STM2215 produced lower levels of effectors of the TTSS-1. STM2215 is a predicted c-di-GMP phosphodiesterase, but lacks identifiable sensor domains. Biochemical analysis of STM2215 determined that it is located in the inner membrane and has c-di-GMP phosphodiesterase activity in vitro dependent on an intact EAL motif. Unlike some previously identified members of this family, STM2215 did not affect motility, was expressed on plates, and in liquid media at late exponential and early stationary phase during growth. Defined mutations in STM2215 revealed that neither the predicted periplasmic domain nor the anchoring of the protein to the inner membrane is necessary for the activity of this protein during infection. However, the EAL domain of STM2215 is required during infection, suggesting that its phosphodiesterase activity is necessary during infection.

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.

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.

Genetic basis for the biosynthesis of methylglucose lipopolysaccharides in Mycobacterium tuberculosis.

Mycobacteria produce two unusual polymethylated polysaccharides, the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) and the 3-O-methylmannose polysaccharides, which have been shown to regulate fatty acid biosynthesis in vitro. A cluster of genes dedicated to the synthesis of MGLP was identified in Mycobacterium tuberculosis and Mycobacterium smegmatis. Overexpression of the putative glycosyltransferase gene Rv3032 in M. smegmatis greatly stimulated MGLP production, whereas the targeted disruption of Rv3032 in M. tuberculosis and that of the putative methyltransferase gene MSMEG2349 in M. smegmatis resulted in a dramatic reduction in the amounts of MGLP synthesized and in the accumulation of precursors of these molecules. Disruption of Rv3032 also led to a significant decrease in the glycogen content of the tubercle bacillus, indicating that the product of this gene is likely to be involved in the elongation of more than one alpha-(1-->4)-glucan in this bacterium. Results thus suggest that Rv3032 encodes the alpha-(1-->4)-glucosyltransferase responsible for the elongation of MGLP, whereas MSMEG2349 encodes the O-methyltransferase required for the 6-O-methylation of these compounds.

Fumonisin B1 exposure and its selective effect on porcine jejunal segment: sphingolipids, glycolipids and trans-epithelial passage disturbance.

Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides, the cause of Fusarium kernel rot in maize. FB(1) is toxic in domestic and laboratory animals, including pigs. This study investigated the effects of a seven-days-exposure of 1.5mg/kg b.w. FB(1) on the porcine intestinal epithelium. Statistically significant increases in the ratio of sphinganine to sphingosine, as well as alterations of the glycolipid distribution were observed in the jejunum. Using a porcine intestinal epithelial cell line (IPEC-1) derived from jejunum and ileum, we tested the effect of FB(1)in vitro in a time- and dose-dependent fashion. A significant increase in sphinganine concentration was observed after 2 days of FB(1) exposure at concentrations >100 microM, or from 6 days of FB(1) exposure at concentration >20 microM. We were also able to show that FB(1) exposure at 200 microM during 16 days increased the intestinal trans-epithelial flux of FB(1). These data indicate that, in pigs, this mycotoxin acts selectively on jejunum cells as follows: (i) FB(1) affects sphingolipid metabolism, as demonstrated by an increase of the amount of free sphingoid bases in a time- and dose-dependent manner, (ii) a depletion of the glycolipids in plasma membranes is observed, and (iii) an increase occurs in the trans-epithelial flux.