Arylamine N-acetyltransferase is required for synthesis of mycolic acids and complex lipids in Mycobacterium bovis BCG and represents a novel drug target

Abstract

Mycolic acids represent a major component of the unique cell wall of mycobacteria. Mycolic acid biosynthesis is inhibited by isoniazid, a key frontline antitubercular drug that is inactivated by mycobacterial and human arylamine N-acetyltransferase (NAT). We show that an in-frame deletion of Mycobacterium bovis BCG nat results in delayed entry into log phase, altered morphology, altered cell wall lipid composition, and increased intracellular killing by macrophages. In particular, deletion of nat perturbs biosynthesis of mycolic acids and their derivatives and increases susceptibility of M. bovis BCG to antibiotics that permeate the cell wall. Phenotypic traits are fully complemented by introduction of Mycobacterium tuberculosis nat. We infer from our findings that NAT is critical to normal mycolic acid synthesis and hence other derivative cell wall components and represents a novel target for antituberculosis therapy. In addition, this is the first report of an endogenous role for NAT in mycobacteria.

Figure 1.

Deleting the nat gene affects the growth of M. bovis BCG Pasteur. (A) Growth of M. bovis BCG and M. bovis BCG Δnat over a 28-d period on solid medium. Colonies of M. bovis BCG are visible by day 21 compared with day 28 for the Δnat strain (magnification, 0.2-fold). (B) Colonies of the Δnat strain (KO) are smaller than the corresponding colonies of the M. bovis BCG (WT) and complemented strain (KO+NAT; magnification, 10-fold). (C) When M. bovis BCG (▴), M. bovis BCG Pasteur Δnat (▪), and nat complemented strains (♦) are grown in liquid culture (7H9 ADC and Tween 80), growth of the M. bovis BCG Δnat strain is altered such that the lag phase is extended and it is restored to the wild-type phenotype when the nat gene is reintroduced. (D) Lysates of cells harvested at mid-log phase were run on SDS-PAGE. In each lane, lysate corresponding to 10 ml culture was loaded. Western blots were developed with specific antibodies against recombinant M. tuberculosis NAT (reference 21) used at a 1:10,000 dilution. Lane 1, pure recombinant NAT (reference 14) as standard (C); lane 2, M. bovis BCG (WT); lanes 3 and 5, Rainbow Molecular Weight Markers (Amersham Biosciences); lane 4, M. bovis BCG Δnat (KO); lane 6, M. bovis BCG Δnat complemented with nat (KO+NAT).

Figure 1.

Deleting the nat gene affects the growth of M. bovis BCG Pasteur. (A) Growth of M. bovis BCG and M. bovis BCG Δnat over a 28-d period on solid medium. Colonies of M. bovis BCG are visible by day 21 compared with day 28 for the Δnat strain (magnification, 0.2-fold). (B) Colonies of the Δnat strain (KO) are smaller than the corresponding colonies of the M. bovis BCG (WT) and complemented strain (KO+NAT; magnification, 10-fold). (C) When M. bovis BCG (▴), M. bovis BCG Pasteur Δnat (▪), and nat complemented strains (♦) are grown in liquid culture (7H9 ADC and Tween 80), growth of the M. bovis BCG Δnat strain is altered such that the lag phase is extended and it is restored to the wild-type phenotype when the nat gene is reintroduced. (D) Lysates of cells harvested at mid-log phase were run on SDS-PAGE. In each lane, lysate corresponding to 10 ml culture was loaded. Western blots were developed with specific antibodies against recombinant M. tuberculosis NAT (reference 21) used at a 1:10,000 dilution. Lane 1, pure recombinant NAT (reference 14) as standard (C); lane 2, M. bovis BCG (WT); lanes 3 and 5, Rainbow Molecular Weight Markers (Amersham Biosciences); lane 4, M. bovis BCG Δnat (KO); lane 6, M. bovis BCG Δnat complemented with nat (KO+NAT).

Figure 1.

Deleting the nat gene affects the growth of M. bovis BCG Pasteur. (A) Growth of M. bovis BCG and M. bovis BCG Δnat over a 28-d period on solid medium. Colonies of M. bovis BCG are visible by day 21 compared with day 28 for the Δnat strain (magnification, 0.2-fold). (B) Colonies of the Δnat strain (KO) are smaller than the corresponding colonies of the M. bovis BCG (WT) and complemented strain (KO+NAT; magnification, 10-fold). (C) When M. bovis BCG (▴), M. bovis BCG Pasteur Δnat (▪), and nat complemented strains (♦) are grown in liquid culture (7H9 ADC and Tween 80), growth of the M. bovis BCG Δnat strain is altered such that the lag phase is extended and it is restored to the wild-type phenotype when the nat gene is reintroduced. (D) Lysates of cells harvested at mid-log phase were run on SDS-PAGE. In each lane, lysate corresponding to 10 ml culture was loaded. Western blots were developed with specific antibodies against recombinant M. tuberculosis NAT (reference 21) used at a 1:10,000 dilution. Lane 1, pure recombinant NAT (reference 14) as standard (C); lane 2, M. bovis BCG (WT); lanes 3 and 5, Rainbow Molecular Weight Markers (Amersham Biosciences); lane 4, M. bovis BCG Δnat (KO); lane 6, M. bovis BCG Δnat complemented with nat (KO+NAT).

Figure 1.

Deleting the nat gene affects the growth of M. bovis BCG Pasteur. (A) Growth of M. bovis BCG and M. bovis BCG Δnat over a 28-d period on solid medium. Colonies of M. bovis BCG are visible by day 21 compared with day 28 for the Δnat strain (magnification, 0.2-fold). (B) Colonies of the Δnat strain (KO) are smaller than the corresponding colonies of the M. bovis BCG (WT) and complemented strain (KO+NAT; magnification, 10-fold). (C) When M. bovis BCG (▴), M. bovis BCG Pasteur Δnat (▪), and nat complemented strains (♦) are grown in liquid culture (7H9 ADC and Tween 80), growth of the M. bovis BCG Δnat strain is altered such that the lag phase is extended and it is restored to the wild-type phenotype when the nat gene is reintroduced. (D) Lysates of cells harvested at mid-log phase were run on SDS-PAGE. In each lane, lysate corresponding to 10 ml culture was loaded. Western blots were developed with specific antibodies against recombinant M. tuberculosis NAT (reference 21) used at a 1:10,000 dilution. Lane 1, pure recombinant NAT (reference 14) as standard (C); lane 2, M. bovis BCG (WT); lanes 3 and 5, Rainbow Molecular Weight Markers (Amersham Biosciences); lane 4, M. bovis BCG Δnat (KO); lane 6, M. bovis BCG Δnat complemented with nat (KO+NAT).

Figure 2.

Morphology and ultrastructure of individual M. bovis BCG Pasteur cells are modified when the nat gene is deleted. Longitudinal (A) and transverse (B) TEM images show that the size of M. bovis BCG is altered on deleting the nat gene. The outer cell wall (arrow), present in M. bovis BCG, is absent in the Δnat strain. SEM (C) also shows the difference in size of the bacilli. Cord formation (arrow) in M. bovis BCG is missing when the nat gene is deleted. Bar is 1 μm for all frames.

Figure 3.

Total lipid and mycolate profile of M. bovis BCG Pasteur is changed when the nat gene is deleted. Analysis of total lipids by two-dimensional TLC shows that tri-acyl glycerol (TAG), MK, and PDIM (A), as well as CF and GMM (B), are present in M. bovis BCG (WT), but missing from the corresponding Δnat BCG strain (NAT KO). All of these complex lipids are restored by complementation with nat (KO+NAT). Separation of mycolates from the same dry weight of delipidated cells by one-dimensional TLC from WT, NATKO, and KO+NAT, shows that the synthesis of mycolate in WT is perturbed by deletion of the nat gene and is fully restored when the M. tuberculosis nat gene is introduced (C). Analyses of fatty acids and multi-acetylated trehaloses (D) and of phospholipids (E) shows very little change between the M. bovis BCG (WT) and Δnat BCG strain (NAT KO). Analyses were performed using the same biomass of the parental and mutant cultures. TAG, tri-acyl glycerol; MK, menaquinone; PDIM, pthiocerol dimycocerosate; CF, cord factor; GMM, glucose monomycolate; MAMES, mycolic acid methyl esters; FAMES, fatty acid methyl esters; MAT, multi-acylated trehaloses; F, Fatty acids; PIMs, phosphatidyl-inositol mannosides; PE, phosphatidyl ethanolamines.

Figure 3.

Total lipid and mycolate profile of M. bovis BCG Pasteur is changed when the nat gene is deleted. Analysis of total lipids by two-dimensional TLC shows that tri-acyl glycerol (TAG), MK, and PDIM (A), as well as CF and GMM (B), are present in M. bovis BCG (WT), but missing from the corresponding Δnat BCG strain (NAT KO). All of these complex lipids are restored by complementation with nat (KO+NAT). Separation of mycolates from the same dry weight of delipidated cells by one-dimensional TLC from WT, NATKO, and KO+NAT, shows that the synthesis of mycolate in WT is perturbed by deletion of the nat gene and is fully restored when the M. tuberculosis nat gene is introduced (C). Analyses of fatty acids and multi-acetylated trehaloses (D) and of phospholipids (E) shows very little change between the M. bovis BCG (WT) and Δnat BCG strain (NAT KO). Analyses were performed using the same biomass of the parental and mutant cultures. TAG, tri-acyl glycerol; MK, menaquinone; PDIM, pthiocerol dimycocerosate; CF, cord factor; GMM, glucose monomycolate; MAMES, mycolic acid methyl esters; FAMES, fatty acid methyl esters; MAT, multi-acylated trehaloses; F, Fatty acids; PIMs, phosphatidyl-inositol mannosides; PE, phosphatidyl ethanolamines.

Figure 4.

Deleting the nat gene affects the intracellular killing of M. bovis BCG Pasteur in macrophages. Infection of mouse macrophage cell line RAW showing that the uptake of M. bovis BCG (WT) and M. bovis BCG Δnat (NATKO) into RAW cells is the same, and there is an equal increase in uptake for both M. bovis BCG and the corresponding Δnat mutant after opsonization (OPS). (A) Fluorimetric assay. (B) FACS^®. Upper right quadrants of B show RAW cells that have taken up FITC-BCG. (C) Intracellular killing assay. The mouse macrophage cell line RAW was infected and samples were taken at the times indicated, plated on agar, and CFUs were counted, showing that M. bovis BCG with and without opsonization can survive and is able to grow within macrophages after 7 d, whereas M. bovis BCG Δnat with and without opsonization are killed between 2 and 72 h after infection. ▴, wild-type strain; ▪, Δnat strain. Solid lines are opsonized and dotted lines are unopsonized.

Figure 4.

Deleting the nat gene affects the intracellular killing of M. bovis BCG Pasteur in macrophages. Infection of mouse macrophage cell line RAW showing that the uptake of M. bovis BCG (WT) and M. bovis BCG Δnat (NATKO) into RAW cells is the same, and there is an equal increase in uptake for both M. bovis BCG and the corresponding Δnat mutant after opsonization (OPS). (A) Fluorimetric assay. (B) FACS^®. Upper right quadrants of B show RAW cells that have taken up FITC-BCG. (C) Intracellular killing assay. The mouse macrophage cell line RAW was infected and samples were taken at the times indicated, plated on agar, and CFUs were counted, showing that M. bovis BCG with and without opsonization can survive and is able to grow within macrophages after 7 d, whereas M. bovis BCG Δnat with and without opsonization are killed between 2 and 72 h after infection. ▴, wild-type strain; ▪, Δnat strain. Solid lines are opsonized and dotted lines are unopsonized.

Figure 4.

Deleting the nat gene affects the intracellular killing of M. bovis BCG Pasteur in macrophages. Infection of mouse macrophage cell line RAW showing that the uptake of M. bovis BCG (WT) and M. bovis BCG Δnat (NATKO) into RAW cells is the same, and there is an equal increase in uptake for both M. bovis BCG and the corresponding Δnat mutant after opsonization (OPS). (A) Fluorimetric assay. (B) FACS^®. Upper right quadrants of B show RAW cells that have taken up FITC-BCG. (C) Intracellular killing assay. The mouse macrophage cell line RAW was infected and samples were taken at the times indicated, plated on agar, and CFUs were counted, showing that M. bovis BCG with and without opsonization can survive and is able to grow within macrophages after 7 d, whereas M. bovis BCG Δnat with and without opsonization are killed between 2 and 72 h after infection. ▴, wild-type strain; ▪, Δnat strain. Solid lines are opsonized and dotted lines are unopsonized.

Figure 5.

Deleting nat affects sensitivity of M. bovis BCG Pasteur to INH. Mid-log phase cultures of M. bovis BCG (▴), M. bovis BCG Δnat (▪), and M. bovis BCG Δnat complemented with nat (♦) were grown in the presence of differing concentrations of INH. The growth was determined after 4 d and is expressed relative to the corresponding strain cultured without INH. Mean values of three determinations are shown and the SD is within the symbol.