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The synthesis of a panel of 5-deoxy-5-methylthio-xylofuranose (MTX)- and 5-deoxy-5-methylsulfoxy-xylofuranose-containing disaccharides was described. Two of the synthesized disaccharides, when tested in assays of cytokine induction did not lead to production of TNF-alpha however, both showed modest inhibitory properties when these cytokines were induced.

Author(s): Peter van den Elzen [1]; Salil Garg [2]; Luis León [2]; Manfred Brigl [2]; Elizabeth A. Leadbetter [2]; Jenny E. Gumperz [2]; Chris C. Dascher [2]; Tan-Yun Cheng [2]; [...]

The lipid-rich Corynebacterianeae, to which Corynebacterium glutamicum and Mycobacterium species belong, produce both fatty acids and mycolic acids. Compared with most other bacteria, C. glutamicum possesses two fatty acid synthases, encoded by fasA (8907 kb; FAS-IA) and fasB (8988 kb; FAS-IB). Here, it was shown by mutational analyses that fasA is essential but fasB is not. However, in a fasA background, the fasB mutation results in a slightly reduced growth yield, L-glutamate production is increased, and comparative lipid analysis suggests that in vivo FAS-IB is active primarily to supply palmitate. Transcript quantifications revealed that the fasB transcript contributes 32% to both fas transcripts during growth on glucose, affirmative for fasB expression, and that fasB is subordinate to fasA. The fasA transcript is downregulated by 8.3-fold during growth on acetate as compared with glucose. The lipid analyses also demonstrate that cells grown on propionate produce a number of uneven fatty acids (e.g. 15 : 0, 17 : 0, 17 : 1), which are not present in cells grown on glucose or acetate, suggesting that fatty acid synthase in vivo may also use propionyl-CoA as the priming unit in fatty acid synthesis. The fatty acid auxotrophic fasAB double mutant was used to determine the suggested incorporation of fatty acids into mycolic acids. Supplementation of this mutant with uniformly labelled [[sup.13]C]oleate and analysis of isolated mycolic acids confirmed that mature mycolic acids in the mutant consist exclusively of two fused [[sup.13]C]oleate molecules. In addition to an altered phospholipid profile, the fasB mutant also exhibits differences in its mycolic acid profile. Taken together, the results show that although FAS-IA is the most relevant fatty acid synthase of C. glutamicum and FAS-IB is supplementary, both synthases are necessary to produce the characteristic lipid environment of this organism.

The azole antifungal drugs econazole and clotrimazole are known cytochrome P450 enzyme inhibitors. This study shows that these drugs are potent inhibitors of mycobacterial growth and are more effective against Mycobacterium smegmatis than isoniazid and ethionamide, two established anti-mycobacterial drugs. Several non-tuberculous mycobacteria, including the pathogenic members of the Mycobacterium avium--intracellulare complex (MAC) and the fast-growing saprophytic organism M. smegmatis, produce an array of serovar-specific (ss) and non-serovar-specific (ns) glycopeptidolipids (GPLs). GPL biosynthesis has been investigated for several years but has still not been fully elucidated. The authors demonstrate here that econazole and clotrimazole inhibit GPL biosynthesis in M. smegmatis. In particular, clotrimazole inhibits all four types of nsGPLs found in M. smegmatis, suggesting an early and common target within their biosynthetic pathway. Altogether, the data suggest that an azole-specific target, most likely a cytochrome P450, may be involved in the hydroxylation of the N-acyl chain in GPL biosynthesis. Azole antifungal drugs and potential derivatives could represent an interesting new range of anti-mycobacterial drugs, especially against opportunistic human pathogens including MAC, M. scrofulaceum, M. peregrinum, M. chelonae and M. abscessus.

Corynebacterium glutamicum is used for the large-scale production of L-glutamate, but the efflux of this amino acid is poorly understood. This study shows that addition of ethambutol (EMB) to growing cultures of C. glutamicum causes L-glutamate efflux at rates of up to 15 nmol [min.sup.-1] [(mg dry wt).sup.-1], whereas in the absence of EMB, no efflux occurs. EMB is used for the treatment of Mycobacterium tuberculosis, and at a molecular level it targets a series of arabinosyltransferases (EmbCAB). The single arabinosyltransferase-encoding emb gene of C. glutamicum was placed under the control of a Tet repressor (TetR). Experiments with this strain, as well as with an emb-overexpressing strain, coupled with biochemical analyses showed that: (i) emb expression was correlated with L-glutamate efflux, (ii) emb overexpression increased EMB resistance, (iii) EMB caused less arabinan deposition in cell wall arabinogalactan, and (iv) EMB caused a reduced content of cell-wall-bound mycolic acids. Thus EMB addition resulted in a marked disordering of the cell envelope, which was also discernible by examining cellular morphology. In order to further characterize the cellular response to EMB addition, genome-wide expression profiling was performed using DNA microarrays. This identified 76 differentially expressed genes, with 18 of them upregulated more than eightfold. Among these were the cell-wall-related genes ftsE and mepA (encoding a secreted metalloprotease); however, genes of central metabolism were largely absent. Given that an altered lipid composition of the plasma membrane of C. glutamicum can result in L-glutamate efflux, we speculate that major structural alterations of the cell envelope are transmitted to the membrane, which in turn activates an export system, perhaps via increased membrane tension.

The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Mycobacterium boris, both of which carry an insertion in Mb0100 (= M. tuberculosis Rv0097) to reveal a new locus involved in PDIM biosynthesis. The mutations have a polar effect on expression of the downstream genes Mb0101, Mb0102 (fadD10), Mb0103, and Mb0104 (nrp), and Mb0100 is shown to be in an operon comprising these genes and Mb0099. Reverse transcription-PCR analysis shows elevated transcription of genes in the operon upstream from the transposon insertion sites in both mutants. Both mutants have altered colony morphology and do not synthesize PDIMs or glycosylphenol-PDIM. Both mutants are avirulent in a guinea pig model of tuberculosis, and when tested as a vaccine, WAg533 conferred protective immunity against M. bovis infection at least equal to that afforded by M. bovis bacillus Calmette-Guerin.

A form of [alpha]-galactosylceramide, KRN7000, activates CD1d-restricted V[alpha]14-invariant (V[alpha]14i) natural killer (NK) T cells and initiates multiple downstream immune reactions. We report that substituting the C26:0 N-acyl chain of KRN7000 with shorter, unsaturated fatty acids modifies the outcome of V[alpha]14i NKT cell activation. One analogue containing a diunsaturated C20 fatty acid (C20:2) potently induced a T helper type 2-biased cytokine response, with diminished IFN-[gamma] production and reduced V[alpha]14i NKT cell expansion. C20:2 also exhibited less stringent requirements for loading onto CD1d than KRN7000, suggesting a mechanism for the immunomodulatory properties of this lipid. The differential cellular response elicited by this class of V[alpha]14i NKT cell agonists may prove to be useful in immunotherapeutic applications. cytokines | inflammation I autoimmunity | immunoregulation

Research has been conducted on Mycobacterium thermoresistibile. The role of growth temperature in M. thermoresistibile complex lipid composition, mycolic and fatty acids has been investigated and the details are reported.

Research has been conducted on the antibiotic thiolactomycin analogues. The synthesis of these analogues has been carried out and the results indicate that they enhance activity against Mycobacterium tuberculosis H37Rv cells and against mycolic acid biosynthesis in M. smegmatis cell extracts.

Research has been conducted on Mycobacterium smegmatis cell wall assembly. The study of the assembly events has been carried out in vivo via radiolabelling of exponentially growing liquid cultures and the ability of M. smegmatis to take up radioactive N-acetylglucosamine from the culture medium and incorporate it into peptidoglycan has been investigated.

Research has been conducted on inositol monophosphatase, an enzyme in the biosynthesis of myo-inositol from insitol-1-phosphate. The activity of this monophosphatase has been investigated and the results of expression, purification and biochemical characterization of Mycobacterium tuberculosis SuhB which possesses high homology degree with human inositol monophosphatase are described.

Author(s): D. Branch Moody (corresponding author) [1]; Timo Ulrichs [1, 2]; Walter Mühlecker [3]; David C. Young [3]; Sudagar S. Gurcha [4]; Ethan Grant [1]; Jean-Pierre Rosat [1]; Michael B. [...]