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Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1β/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1β processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1β expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.

The mycobacterial cell wall consists of a core composed of peptidoglycan linked to the heteropolysaccharide arabinogalactan, which in turn is attached to mycolic acids. A variety of free lipids complements the mycolyl residues, whereas phosphatidylinositol mannosides (PIMs), lipoarabinomannan and proteins are interspersed in this framework. As a consequence, the cell envelope is extremely rich in lipids and early work has shown that the lipid content may vary with environmental conditions. To extend these studies, the influence of growth temperature on cell envelope components in Mycobacterium thermoresistibile, a temperature-resistant mycobacterial species, was investigated. Mycolic acid synthesis was reduced at 55 degrees C compared to 37 degrees C and the production of fatty acids, presumably precursors of mycolic acids, was increased. Since fatty acids are elongated by the type II fatty acid synthase complex and consequently by a mycobacterial beta-ketoacyl acyl carrier protein synthase (KasA), leading to mycolic acids, the expression level of KasA was analysed by Western blotting. KasA expression was significantly decreased at 55 degrees C over 37 degrees C. Important changes in the mycolic acid composition were observed and characterized by reduced levels of cyclopropanation and the concomitant accumulation of the cis-olefin derivatives. In addition, striking differences involved in complex lipid composition, including acylated trehaloses and trehalose dimycolate (TDM) were also observed. At 55 degrees C, M. thermoresistibile produced less TDM than at 37 degrees C, which could be explained by the down-regulation of antigen 85 (Ag85) expression as shown by Western blotting. The Ag85 complex represents a family of proteins known to catalyse the transfer of mycolates to trehalose, thereby generating TDM. Furthermore, at 55 degrees C the level of phosphatidyl-inositol hexamannoside (PIM(6)) synthesis, but not that of other PIM species, was dramatically reduced. This observation could be correlated to a decrease of mannosyltransferase activity associated with membranes prepared from cells grown at 55 degrees C as compared to 37 degrees C. Altogether, this study suggests that mycobacteria are capable of inducing important cell-wall changes in response to temperature variations, which may represent a strategy developed by the bacteria to adapt to environmental changes.

Synthetic peptides derived from a 45-kDa glycoprotein antigen of Mycobacterium tuberculosis were shown to function as glycosyltransferase acceptors for mannose residues in a mannosyltransferase cell-free assay. The mannosyltransferase activity was localized within both isolated membranes and a P60 cell wall fraction prepared from the rapidly growing mycobacterial strain, Mycobacterium smegmatis. Incorporation of radiolabel from GDP-[(14)C]mannose was inhibited by the addition of amphomycin, indicating that the glycosyl donor for the peptide acceptors was a member of the mycobacterial polyprenol-P-mannose (PPM) family of activated glycosyl donors. Furthermore, a direct demonstration of transfer from the in situ generated PP[(14)C]Ms was also demonstrated. It was also found that the enzyme activity was sensitive to changes in overall peptide length and amino acid composition. Because glycoproteins are present on the mycobacterial cell surface and are available for interaction with host cells during infection, protein glycosyltransferases may provide novel drug targets. The development of a cell-free mannosyltransferase assay will now facilitate the cloning and biochemical characterisation of the relevant enzymes from M. tuberculosis.

The mycobacterial cell wall is a potential target for new drug development. Herein we report the preparation and activity of several n-octyl-5-(alpha-D-arabinofuranosyl)-beta-D-galactofuranoside derivatives. A cell-free assay system has been utilized for determination of the ability of disaccharide analogues to act as arabinosyltransferase acceptors using [14C]-DPA as the glycosyl donor. In addition, in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). One of these disaccharides showed moderate activity against MTB. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to a totally deprotected disaccharide that more closely resembles the natural substrates in cell wall biosynthesis.

Extracts from forty-three plant species were selected on account of reported traditional uses for the treatment of TB and/or leprosy. These were assayed for antimycobacterial activities. A simple in vitro screening assay was employed using two model species of mycobacteria, M. aurum and M. smegmatis. Crude methanolic extracts from three of the plants, C. mukul, P. corylifolia and S. canadensis, were found to have significant antimycobacterial activity against M. aurum only (MIC=62.5 microg/ml). Bioassay guided fractionation led to the isolation of two known benzophenanthridine alkaloids, sanguinarine (1) and chelerythrine (2), from the roots S. canadensis and the known phenolic meroterpene, bakuchiol (3) from the seeds of P. corylifolia. The fractionation of the resin of C. mukul lead to a decrease in antimycobacterial activity and hence further work was not pursued. Compound (2) was the most active against M. aurum and M. smegmatis (IC(50)=7.30 microg/ml [19.02 microM] and 29.0 microg/ml [75.56 microM], respectively). M. aurum was the most susceptible organism to all three compounds. No significant difference in antimycobacterial activity was observed when the two alkaloids were tested for activity in media of differing pH values. The activities of the pure compounds against M. aurum were comparable with those against M. bovis BCG with compound (2) being the most active (M. bovis BCG, IC(50)=14.3 microg/ml [37.3 microM]). These results support the use of these plants in traditional medicine.

Demonstrates that the polyprenol phosphate mannose synthase Ppm1 binds to Ppm2 from Mycobacterium smegmatis in vivo using a bacterial two-hybrid system. Indication that MsPpm2 is an integral membrane protein and MsPpm1 lacks the characteristic hydrophobic C terminus; Comparison with mammalian dolichol phosphate-mannose synthases.

Abstract: The arabinogalactan (AG) component of the mycobacterial cell wall is an essential branched polysaccharide which tethers mycolic acids (m) to peptidoglycan (P), forming the mAGP complex. Much interest has been focused on the biosynthetic machinery involved in the production of this highly impermeable shield, which is the target for numerous anti-tuberculosis agents. The galactan domain of AG is synthesised via a bifunctional galactofuranosyltransferase (GlfT), which utilises UDP-Galf as its high-energy substrate. However, it has proven difficult to study the protein in its recombinant form due to difficulties in recovering pure soluble protein using standard expression systems. Herein, we describe the effects of glfT co-induction with a range of chaperone proteins, which resulted in an appreciable yield of soluble protein at 5mg/L after a one-step purification procedure. We have shown that this purified enzyme transfers [14C]Galf to a range of both β(1→5) and β(1→6) linked digalactofuranosyl neoglycolipid acceptors with a distinct preference for the latter. Ligand binding studies using intrinsic tryptophan fluorescence have provided supporting evidence for the apparent preference of this enzyme to bind the β(1→6) disaccharide acceptor. However, we could not detect binding or galactofuranosyltransferase activity with an n-octyl β-d-Gal-(1→4)-α-l-Rha acceptor, which mimics the reducing terminus of galactan in the mycobacterial cell wall. Conversely, after an extensive bioinformatics analysis of the H37Rv genome, further cloning, expression and functional analysis of the Rv3792 open reading frame indicates that this protein affords galactofuranosyltransferase activity against such an acceptor and paves the way for a better understanding of galactan biosynthesis in Mycobacterium tuberculosis. [Copyright &y& Elsevier]

Examines the azole antifungal to be potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in Mycobacteria and Streptomycetes. Identification on the genome sequence of Mycobacterium tuberculosis; Discussion on the physiological roles of enzymes; Efficacy of econazole and clotrimazole against M. Smegmatis.