Methods of diagnosing multidrug resistant tuberculosis

#Not applicable.

#The World Health Organization (“WHO”) estimates that as much as one-third of the world's population is infected with tuberculosis. In 1998, the latest year for which estimates are available, Mycobacterium tuberculosis (“MTb”) infected 7.25 million people and resulted in 2.9 million fatalities (Farmer, P. et al., Int J Tuberc Lung Dis 2:869 (1998)). Underlying these statistics is an emerging epidemic of multiple drug-resistant (“MDR”) tuberculosis that severely undermines control efforts and is transmitted indiscriminately across national borders (Viskum, K. et al., Int J Tuberc Lung Dis 1:299 (1997); Bass, J. B. et al., Am J Respir Crit Care Med 149:1359 (1994)). Resistance to any of the front-line drugs generally bodes poorly for the patient, who then is committed to a regimen of less active “second-line” therapies. Where multidrug resistance is suspected, the WHO recommends that three or more drugs be administered at the same time, to decrease the chance that the organism will be able to develop resistance to all of the agents.

#One of the most efficacious of the second-line drugs is the thioamide ethionamide (ETA) (Farmer, P. et al., supra). Like the front-line drug, isoniazid (INH), ETA is specific for mycobacteria and is thought to exert a toxic effect on mycolic acid constituents of the cell wall of the bacillus (Rist, N. Adv Tuberc Res 10:69 (1960); Banerjee, A. et al., Science 263:227 (1994)). Current tuberculosis therapies include a large number of “prodrugs” that must be metabolically activated to manifest their toxicity upon specific cellular targets (Barry, C. B., III et al., Biochem Pharm 59:221 (2000)). The best characterized example of this is the activation of INH by the catalase-peroxidase KatG, generating a reactive form that then inactivates enzymes involved in mycolic acid biosynthesis (Slayden, R. A. et al., Microbes and Infection (2000) (in press); Heym, B. et al., Tubercle Lung Dis 79:191 (1999)). The majority of clinically observed INH resistance is associated with the loss of this activating ability by the bacillus (Musser, J. M., Clin Microbiol Rev 8:496 (1995)), but such strains typically retain their sensitivity toward ETA, suggesting that ETA activation requires a different enzyme than KatG (Rist, N., Adv. Tub. Res. 10, 69 (1960)).

#FIG. 1. In vivo production of (2-ethyl-pyridin-4-yl)methanol (5) from ETA by whole cells of MTb.

#FIG. 1A. Metabolism of radiolabeled ETA by MTb. Lanes a-h correspond to sequential supernatant samples taken at times: 0.2, 0.25, 0.75, 1.5, 5.0, 8.5, and 25 hours, respectively. Lane i represents media autooxidation following 25 hr of incubation without bacterial cells. These metabolites correspond to ETA S-oxide (2), ETA nitrile (3) and ETA amide (4)

#FIG. 1B. Cell associated radioactivity counts graphed against time. “DPM,” disintergrations per minute.

#Introduction

#It has now been discovered that two of the putative genes of M. tuberculosis, Rv3854c and Rv3855, regulate the susceptibility of M. tuberculosis to the major second-line drug, ethionamide (“ETA”), used to treat tuberculosis. Specifically, it has now been discovered that the gene currently known as Rv3854c is a monooxygenase. Further, it has now been discovered that this gene confers upon Mycobacteria the ability to activate thioamide and thiocarbonyl drugs from their prodrug form to their active drug form. When the tuberculosis genome was sequenced and analyzed in 1998, the gene was considered to bear homology to a bacterial monooxygenase, but was sufficiently different to be classified as a separate, unknown family. Moreover, its substrate was unknown.

#It has now further been discovered that the gene currently known as Rv3855 is a regulator of expression of the monooxygenase encoded by Rv3854c, and can repress its expression. In recognition of the discovery of the functions of these genes, we have renamed Rv3854c and Rv3855 as EtaA and EtaR, respectively.