ACCase 6 is the essential acetyl-CoA carboxylase involved in fatty acid and mycolic acid biosynthesis in mycobacteria

Abstract

Mycolic acids are essential for the survival, virulence and antibiotic resistance of the human pathogen Mycobacterium tuberculosis. Inhibitors of mycolic acid biosynthesis, such as isoniazid and ethionamide, have been used as efficient drugs for the treatment of tuberculosis. However, the increase in cases of multidrug-resistant tuberculosis has prompted a search for new targets and agents that could also affect synthesis of mycolic acids. In mycobacteria, the acyl-CoA carboxylases (ACCases) provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I products by the FAS II complex to produce meromycolic acids. By generating a conditional mutant in the accD6 gene of Mycobacterium smegmatis, we demonstrated that AccD6 is the essential carboxyltransferase component of the ACCase 6 enzyme complex implicated in the biosynthesis of malonyl-CoA, the substrate of the two FAS enzymes of Mycobacterium species. Based on the conserved structure of the AccD5 and AccD6 active sites we screened several inhibitors of AccD5 as potential inhibitors of AccD6 and found that the ligand NCI-172033 was capable of inhibiting AccD6 with an IC(50) of 8 microM. The compound showed bactericidal activity against several pathogenic Mycobacterium species by producing a strong inhibition of both fatty acid and mycolic acid biosynthesis at minimal inhibitory concentrations. Overexpression of accD6 in M. smegmatis conferred resistance to NCI-172033, confirming AccD6 as the main target of the inhibitor. These results define the biological role of a key ACCase in the biosynthesis of membrane and cell envelope fatty acids, and provide a new target, AccD6, for rational development of novel anti-mycobacterial drugs.

Fig. 1.

Allelic exchange of the accD6 locus of M. smegmatis. (a–c) Genetic organization, partial restriction map and expected hybridization profiles of the accD6 chromosomal region in (a) the wild-type strain mc²155, (b) the single crossover strain D6SCO1 and (c) the M. smegmatis D6DCO2 conditional mutant. Oligonucleotides used to confirm single and double crossover events by PCR are indicated. (d) Southern blot analysis of M. smegmatis accD6 mutants. Three mutants were picked at random (1, 2 and 3); chromosomal DNA was digested with EcoRV and probed for hybridization with a labelled 666 bp fragment corresponding to the 5′ region of kasB. M. smegmatis mc²155 DNA was included as a control (wt). Molecular masses are indicated in kb.

Fig. 2.

Growth characteristics and lipid composition of the accD6 conditional mutant D6DCO2 incubated at 30 and 42 °C. (a) Growth curves of strains mc²155/pCG76 (•, ○) and D6DCO2 (▾, ▵) incubated at 30 °C (black symbols) and 42 °C (white symbols). Saturated cultures grown at 30 °C were diluted in fresh 7H9 medium supplemented with ADS to an OD₆₀₀ of 0.1 and further incubated at 30 or 42 °C. The arrow indicates growth arrest of D6DCO2. (b) At different time points, the number of viable cells of D6DCO2 in the cultures grown at 30 °C (•) and 42 °C (○) was evaluated by plating serial dilutions onto LB plates at 30 °C. (c) Saturated cultures of strains mc²155/pCG76 and D6DCO2 were diluted in fresh 7H9 medium and incubated at 42 °C. At −3, 1, 2 and 4 h before or after D6DCO2 stopped growing (see a), aliquots from both cultures containing the same number of cells were labelled with [¹⁴C]acetate for 1 h at 42 °C. One half of each sample was used to study the fatty acid and mycolic acid compositions of the strains by TLC (solvent system: hexane/ethyl acetate, 10 : 1).

Fig. 3.

(a) Loss of acetyl-CoA carboxylase activity in the D6DCO2 mutant strain at restrictive growth temperature. Cell-free extracts were prepared from strains mc²155/pCG76 and D6DCO2 growing at 42 °C (−3, 2, and 4 h before or after D6DCO2 stopped growing), and acetyl-CoA carboxylase activity was determined for each sample as indicated in Methods. Results are the means of three independent experiments. (b) Determination of metabolic activity by incorporation of [³H]leucine. At different time points aliquots of mc²155/pCG76 and D6DCO2 cultures growing at 42 °C were labelled for 1 h with [³H]leucine and the radioactivity incorporated into the cells was measured as indicated in Methods. The results were normalized by OD₆₀₀ and are the means of three independent experiments.

Fig. 4.

(a) Chemical structure of NCI-172033. (b) IC₅₀ determination. ACCase activity of AccD5 (•) and AccD6 (▾) was measured using the pyruvate-kinase-coupled enzyme assay at different concentrations of NCI-172033 and K_m concentration of the acyl-CoA substrate. IC₅₀ values of 8 μM for the ACCase 6 complex and 78 μM for the ACCase 5 complex were determined. Results are the means of at least four independent experiments with a standard error that was within ±5 % of the mean.

Fig. 5.

Dose–response effects of NCI-172033 on fatty acid and mycolic acid biosynthesis in M. smegmatis (a) and M. tuberculosis (b). The inhibitory effect on the incorporation of [¹⁴C]acetate was assayed by labelling the individual cultures in the presence of increasing concentrations of NCI-172033 and inhibitory concentrations of isoniazid and cerulenin. The corresponding FAMEs and MAMEs were isolated and analysed by TLC.

Fig. 6.

In vivo effect of NCI-172033 on the ACCase, PCCase and FAS I activities of M. smegmatis. Cultures of M. smegmatis were incubated in the presence of 100 μM NCI-172033 and the relative acetyl- and propionyl-CoA-dependent carboxylases and FAS I activities determined in the cell-free extracts. Cerulenin, a known inhibitor of the β-ketoacyl synthase domain in type I synthases, was used as a control.