The thiourea isoxyl (thiocarlide; 4,4-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 g/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a ⌬9-stearoyl desaturase as the drug target. Development of a cell-free assay for ⌬9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of ⌬9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased ⌬9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound ⌬9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.The prevalence of tuberculosis, particularly in concert with human immunodeficiency virus infection and AIDS, has been well documented (1). An equally serious public health problem is increasing multi-drug-resistant tuberculosis (2). At present only a few alternative chemotherapeutic regimens are available, resulting in poor therapeutic outcomes and high mortality rates among multi-drug-resistant tuberculosis patients (3). There is an urgent need to develop new effective antituberculosis drugs with bactericidal mechanisms different from those of the presently available agents.It is prudent to re-examine drugs that were formerly deemed effective against tuberculosis. Isoxyl (ISO) 1 (thiocarlide) (Fig. 1) is a thiourea derivative that was successfully used in the 1960s to treat tuberculosis (4 -7). Recently, ISO was shown to have considerable antimycobacterial activity in vitro and to be effective against various clinical isolates of multidrug-resistant strains of Mycobacterium tuberculosis in the range of 1-10 g/ml (8). An early note reported that ISO, like isoniazid (INH) and ethionamide (ETH), strongly inhibits the synthesis of mycolic acids (9), a result since confirmed with the demonstration that all types of mycolic acids are affected (8). In addition it was noted that ISO also inhibited shorter chain fatty acid synthesis (8 -11), suggesting inhibitory effects dif...

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg 2؉ ions and exhibits optimal activity between pH 7.5 and 7.9; the K m for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 M, and the K m for NADPH was 29.7 M. The specificity constant of Rv2780c in the forward direction is 1.5 ؋ 10 6 M ؊1 min ؊1 , and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.

The peptidoglycan of Mycobacterium spp. reportedly has some unique features, including the occurrence of N-glycolylmuramic rather than N-acetylmuramic acid. However, very little is known of the actual biosynthesis of mycobacterial peptidoglycan, including the extent and origin of N glycolylation. In the present work, we have isolated and analyzed muramic acid residues located in peptidoglycan and UDP-linked precursors of peptidoglycan from Mycobacterium tuberculosis and Mycobacterium smegmatis. The muramic acid residues isolated from the mature peptidoglycan of both species were shown to be a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported. The isolated UDP-linked N-acylmuramyl-pentapeptide precursor molecules also contain a mixture of N-acetyl and N-glycolyl muramyl residues in apparent contrast to previous observations in which the precursors isolated after treatment with D The peptidoglycan of Mycobacterium spp. is classified as A1␥, unmodified peptidoglycan, as is that of Escherichia coli and many other bacterial species (32). However, the peptidoglycan of mycobacteria contains a variety of reported modifications including, invariably, an N-glycolyl (NGlyc) instead of an N-acetyl (NAc) function on the muramic acid (Mur), amidation of the carboxylic acids, and additional glycine or serine residues (18,20). The peptidoglycan biosynthetic pathway of E. coli has been well studied (34,35), and the mycobacterial pathway is assumed to be the same, based on limited biochemical analysis of some years ago (25, 28, 33) and more recent comparative genomics (2, 5, 6, 21). Analysis of the various precursors of peptidoglycan biosynthesis in Mycobacterium spp. could provide important information on the biosynthetic origin of these modifications and the notable refractoriness of Mycobacterium tuberculosis to ␤-lactam antibiotics and help in the search for alternative drug regimens for the treatment of multiple-drug-resistant forms of tuberculosis (10, 16).UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine (UDP-MurNAc-pentapeptide) is the final cytosolic precursor of peptidoglycan biosynthesis in E. coli. Because of the relatively low abundance of these precursors in untreated cells, drug treatment was used in order to obtain sufficient amounts for chemical characterization in mycobacterial studies. An incomplete precursor, UDP-Nglycolylmuramyl-L-alanyl-D-glutamyl-diaminopimelic acid (UDP-MurNGlyc-tripeptide) was isolated and analyzed from M. tuberculosis and Mycobacterium phlei following accumulation after treatment with D-cycloserine (28, 33). During a comparative study, we observed that the muramic acid residues isolated from mature peptidoglycan of M. tuberculosis and Mycobacterium smegmatis were a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported (4,19,20). Since the nucleotide-linked precursors should reflect the nature of mature peptidoglycan, we hypothesized that the...

The histone chaperone FACT functions by tethering partial components of the nucleosome, thereby assisting nucleosome disassembly and reassembly during transcription.

Human FACT (hFACT) is a conserved histone chaperone that was originally described as a transcription elongation factor with potential nucleosome assembly functions. Here we show that FACT facilitates tetrasome assembly and H2A-H2B deposition to form hexasomes and nucleosomes. In the process, FACT tethers components of the nucleosome through interactions with H2A-H2B, resulting in a defined intermediate complex comprised of FACT, a histone hexamer and DNA. Free DNA extending from the tetrasome then competes FACT off H2A-H2B, thereby promoting hexasome and nucleosome formation. Our studies provide mechanistic insight into how FACT may stabilize partial nucleosome structures during transcription or nucleosome assembly, seemingly facilitating nucleosome disassembly and nucleosome assembly.