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...
A number of nucleic acid amplification assays (NAAs) have been employed to detect tubercle bacilli in clinical specimens for tuberculosis (TB) diagnosis. Among these, loop-mediated isothermal amplification (LAMP) is an NAA possessing superior isothermal reaction characteristics. In the present study, a set of six specific primers targeting the Mycobacterium tuberculosis 16S rRNA gene with high sensitivity was selected and a LAMP system (MTB-LAMP) was developed. Using this system, a total of 200 sputum samples from Nepalese patients were investigated. The sensitivity of MTB-LAMP in culture-positive samples was 100 % (96/96), and the specificity in culture-negative samples was 94.2 % (98/104, 95 % confidence interval 90.5-97.9 %). The positive and negative predictive values of MTB-LAMP were 94.1 and 100 %, respectively. These results indicate that this MTB-LAMP method may prove to be a powerful tool for the early diagnosis of TB.
Isoxyl (ISO), a thiourea (thiocarlide; 4,4′-diisoamyloxythiocarbanilide), demonstrated potent activity againstMycobacterium tuberculosis H37Rv (MIC, 2.5 μg/ml),Mycobacterium bovis BCG (MIC, 0.5 μg/ml),Mycobacterium avium (MIC, 2.0 μg/ml), andMycobacterium aurum A+ (MIC, 2.0 μg/ml), resulting in complete inhibition of mycobacteria grown on solid media. Importantly, a panel of clinical isolates of M. tuberculosis from different geographical areas with various drug resistance patterns were all sensitive to ISO in the range of 1 to 10 μg/ml. In a murine macrophage model, ISO exhibited bactericidal killing of viable intracellular M. tuberculosis in a dose-dependent manner (0.05 to 2.50 μg/ml). The selective action of ISO on mycolic acid synthesis was studied through the use of [1,2-14C]acetate labeling of M. tuberculosis H37Rv, M. bovisBCG, and M. aurum A+. At its MIC for M. tuberculosis, ISO inhibited the synthesis of both fatty acids and mycolic acids (α-mycolates by 91.6%, methoxymycolates by 94.3%, and ketomycolates by 91.1%); at its MIC in M. bovis BCG, ISO inhibited the synthesis of α-mycolates by 87.2% and that of ketomycolates by 88.5%; and the corresponding inhibitions for M. aurum A+ were 87.1% for α-mycolates, 87.2% for ketomycolates, and 86.5% for the wax-ester mycolates. A comparison with isoniazid (INH) and ethionamide (ETH) demonstrated marked similarity in action, i.e., inhibition of the synthesis of all kinds of mycolic acids. However, unlike INH and ETH, ISO also inhibited the synthesis of shorter-chain fatty acids. ISO showed no acute toxicity against primary macrophage cell cultures as demonstrated by diminution of redox activity. A homologous series of ISO derivatives were synthesized. Most derivatives were as effective or more effective than the parent compound in the agar proportion assay. Thus, these thioureas, like INH and ETH, specifically inhibit mycolic acid synthesis and show promise in counteracting a wide variety of drug-sensitive and -resistant strains of M. tuberculosis.
An RNA-based assay is an additional molecular tool for leprosy diagnosis and determination of the viability of leprosy bacilli. To simplify RNA detection, a one-step reverse transcriptase PCR (RT-PCR) was established and evaluated. RNA and DNA could be isolated simultaneously. With the use of Mycobacterium leprae-specific primers targeting a 171-bp fragment of the M. leprae 16S RNA gene, RT-PCR resulted in detection of M. leprae in both slit skin smears and skin biopsy specimens. To enhance the positive signal, a digoxigenin-labeled DNA was developed, and successfully detected the amplified RT-PCR product. The method is sensitive, as it could detect one leprosy bacillus. When it was used directly on skin specimens collected from leprosy patients, 34 of 36 multibacillary (MB) and 13 of 24 paucibacillary (PB) cases showed positive results. The assay was also effective in monitoring bacterial clearance in leprosy patients during chemotherapy; after treatment with the multidrug therapy for 6 months, resulting in bacterial clearance, 16 of 36 MB patients and three of 24 PB patients tested were still positive for the 16S rRNA gene of M. leprae, suggesting the advisability of a more prolonged treatment course. This form of RT-PCR is of value in terms of simplicity and sensitivity in identifying M. leprae in routine skin specimens, especially when acid-fast bacilli are not discernable.
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