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Arabinosyltransferases are a family of membrane-bound glycosyltransferases involved in the biosynthesis of the arabinan segment of two key glycoconjugates, arabinogalactan and lipoarabinomannan, in the mycobacterial cell wall. All arabinosyl-transferases identified have been found to be essential for the growth of Mycobcterium tuberculosis and are potential targets for developing new antituberculosis drugs. Technical bottlenecks in designing enzyme assays for screening for inhibitors of these enzymes are (1) the enzymes are membrane proteins and refractory to isolation; and (2) the sole arabinose donor, decaprenylphosphoryl-d-arabinofuranose is sparingly produced and difficult to isolate, and commercial substrates are not available. In this study, we have synthesized several analogues of decaprenylphosphoryl-d-arabinofuranose by varying the chain length and investigated their arabinofuranose (Araf) donating capacity. In parallel, an essential arabinosyltransferase (AftC), an enzyme that introduces α-(1→3) branch points in the internal arabinan domain in both arabinogalactan and lipoarabinomannan synthesis, has been expressed, solubilized, and purified for the first time. More importantly, it has been shown that the AftC is active only when reconstituted in a proteoliposome using mycobacterial phospholipids and has a preference for diacylated phosphatidylinositoldimannoside (Ac2PIM2), a major cell wall associated glycolipid. α-(1→3) branched arabinans were generated when AftC–liposome complex was used in assays with the (Z,Z)-farnesylphosphoryl d-arabinose linear α-d-Araf-(1→5)3–5 oligosaccharide acceptors and not with the acceptor that had a α-(1→3) branch point preintroduced.

Isoxyl (ISO), a thiourea derivative that was successfully used for the clinical treatment of tuberculosis during the 1960s, is an inhibitor of the synthesis of oleic and mycolic acids in Mycobacterium tuberculosis. Its effect on oleic acid synthesis has been shown to be attributable to its inhibitory activity on the stearoylcoenzyme A desaturase DesA3, but its enzymatic target(s) in the mycolic acid pathway remains to be identified. With the goal of elucidating the mode of action of ISO, we have isolated a number of spontaneous ISO-resistant mutants of M. tuberculosis and undertaken their genotypic characterization. We report here the characterization of a subset of these strains carrying mutations in the monooxygenase gene ethA. Through complementation studies, we demonstrate for the first time that the EthA-mediated oxidation of ISO is absolutely required for this prodrug to inhibit its lethal enzymatic target(s) in M. tuberculosis. An analysis of the metabolites resulting from the in vitro transformation of ISO by purified EthA revealed the occurrence of a formimidamide allowing the formulation of an activation pathway in which the oxidation of ISO catalyzed by EthA is followed by chemical transformations involving extrusion or elimination and, finally, hydrolysis.Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), claims about 1.7 million lives annually, and the global number of TB cases is still rising at a rate of 0.6% per year (28). Approximately 2% of new TB cases in the world are attributed to multidrug-resistant strains, defined as M. tuberculosis isolates resistant to at least isoniazid and rifampin, the two most powerful anti-TB drugs. However, multidrug resistance rates in some countries, particularly in the former Soviet Union, Asia, the Dominican Republic, and Argentina, are much higher and may reach more than 22% of all isolates (16). Moreover, extensively drug-resistant strains that are resistant to three or more of the six classes of second-line drugs in addition to rifampin and isoniazid have been reported in all regions of the world (5), raising the possibility of future epidemics of virtually untreatable forms of TB. The continuing rise in disease incidence, the problem of drug resistance, and the need to reduce treatment duration has prompted research on new drug developments (11). In this light, the reexamination of isoxyl (ISO) (4,4Ј-diisoamyloxydiphenylthiourea; 4,4Ј-diisoamyloxythiocarbanilide; thiocarlide), a thiourea derivative that was successfully used for the clinical treatment of TB during the 1960s, may prove useful in identifying new therapeutic targets and in developing new therapeutic agents with greater potency and more desirable features than earlier thioureas (1,14,21,(23)(24)).An early article reported that ISO inhibits the synthesis of mycolic acids and free fatty acids in Mycobacterium bovis BCG (27). We later demonstrated that ISO displays potent activity against other slow-and fast-growing species of Mycobacterium, including multidrug-resistant clinic...

Abstractp-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor α, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the proinflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.

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