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Mycobacteria: Genomics, BACs and Resistance
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Antibiotics and chemo-therapeutics involved in TB treatment strategies:

* RIFAMPIN (RIF) (binds to the beta subunit of RNA polymerase, inhibiting transcription initiation)
genes involved in resistance: rpoB (encoding a beta subunit of RNA polymerase)

* ISONIAZID (INH) (activated form inhibits mycolic acid biosynthesis)
genes involved in resistance:
katG (encoding a catalase-peroxydase needed for activation)
inhA ( encoding a enoyl-acyl carrier protein)
kasA (encoding a beta-ketoacyl acyl carrier protein synthase)
possibly involved : ahpc, furA

* ETHIONAMIDE (ETH) (analog of INH inhibits mycolic acid biosynthesis)
genes involved in resistance
inhA ( encoding a enoyl-acyl carrier protein)

* STREPTOMYCIN (STR) (aminocyclitol glycoside antibiotic, binds to 16S rRNA and inhibits translational initiation)
genes involved in resistance:
rrs (16S rRNA gene)
rpsL (encoding ribosomal protein S12)

* PYRAZINAMIDE (PZA) (structual analog of nicotinamide, activated by pyrazinamidase (encoded by pncA) into it's acive form pyrazyinoic acid)
genes involved in resistance:
pncA (encoding a pyrazinamidase)

* ETHAMBUTOL (EMB) (inhibits araninogalactan transfer into the cell wall)
genes involved in resistance:
embB gene in the embCAB operon

* FLUOROQUINOLONES (bind to DNA gyrase and inhibit supercoiling)
Ciprofloxacin (CIP)
Ofloxacin (OFX)

genes involved in resistance: gyrA gene

* KANAMYCIN (KAN), AMIKACIN (AMI), VIOMYCIN (VIO) (aminoglycoside antibiotics that inhibit protein synthesis)
genes involved in resistance: rrs (16S rRNA gene)

* CYCLOSERINE (D-cycloserine = analog of D-alanine that inhibits cell wall synthesis)
genes involved in resistance: ?? alrA, D-alanine racemase gene

Antibiotic resistance and oxidative stress response of mycobacteria

Resistance to antimycobacterial agents. Isoniazid (INH) is a potent tuberculocidal agent that is transformed into toxic acyl radicals by the peroxidative activity of catalase-peroxidase, KatG, the first virulence factor identified in mycobacteria. Important progress has been made in our understanding of this enzymatic mechanism as a result of extensive structure-function studies and protein engineering. The Ser315Thr mutation that is commonly found in the katG gene of INH-resistant clinical isolates leads to a two-fold reduction in both catalase and peroxidase activities but more than ten-fold decrease in the ability of the variant enzyme to produce the INH-radicals that inhibit mycolic acid synthesis. The prevalence of this mutation that confers high level drug resistance suggests that it has been selected because the residual catalase activity confers extensive protection against the oxidative burst of host macrophages. (Pym, A.S., Saint-Joanis, B., and Cole S.T. (2002) Effect of katGMutations on the Virulence of Mycobacterium tuberculosisand the Implication for Transmission in Humans. Infect Immun. 70:4955-4960, pdf)
As part of the M. tuberculosis structural genomics programme, the crystal structures of KatG and the functionally related alkyl hydroperoxide reductase, AhpC are being solved at the Unité de Biochimie Structurale

Although the molecular basis of INH-resistance can be explained in most strains, a small percentage contain mutations to as yet unidentified genes that result in clinically-significant resistance levels. One of the potential candidates that we are studying is oxyS, a gene that encodes a regulatory protein belonging to the LysR family. An oxyS knock-out mutant of M. tuberculosis has been constructed by homologous recombination and preliminary results show that the mutant has altered levels of expression of enzymes involved in resisting oxidative stress. The virulence of the mutant will be studied shortly.

Further reading:
Domenech, P., Honore, N., Heym, B. and Cole, S.T. (2001) Role of OxyS of Mycobacterium tuberculosis in oxidative stress: overexpression confers increased sensitivity to organic hydroperoxides. Microbes Infect 3: 713-21 (pdf)

Pym, A.S., Domenech, P., Honore, N., Song, J., Deretic, V., and Cole, S.T. (2001) Regulation of catalase-peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis. Mol Microbiol. 40:879-889(HTML).

Pym, A.S. and Cole, S.T. (2002) Tuberculosis chemotherapy - from conception to genomics. In Bacterial Resistance to Antimicrobials: Mechanisms, Genetics, Medical Practice and Public Health. Wax, R., Lewis, K., Salyers, A. and Taber, H. (eds). New York: Marcel Dekker, Inc., pp. 355-403.

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