Phosphorylation of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA regulates mycolic acid biosynthesis

Abstract

Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential beta-ketoacyl-acyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr(191) being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr(191) alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

FIGURE 1.

Mycolic acid biosynthetic pathway. The malonyl-CoA:ACP transacylase mtFabD converts malonyl-CoA into malonyl-ACP, providing the elongation building blocks for the FAS-II. Cycles of elongation are initiated by the condensation of the FAS-I acyl-CoA products with malonyl-ACP, a reaction catalyzed by the β-ketoacyl-ACP synthase mtFabH. The second step in the elongation cycle is performed by the NADPH-dependent β-ketoacyl-ACP reductase MabA, generating a β-hydroxyacyl-ACP intermediate, which is subsequently dehydrated by the β-hydroxyacyl-ACP dehydratase HadABC to form a trans-Δ²-enoyl-ACP. The final step in the elongation is carried out by the NADH-dependent enoyl-ACP reductase InhA. Subsequent rounds of elongation are initiated by the elongation condensing enzymes KasA and KasB, giving raise to meromycolic acids, which are condensed with C₂₆ acyl-CoAs by the termination condensing enzyme Pks13 to form mycolic acids.

FIGURE 2.

In vitro phosphorylation of MabA. A, phosphorylation of MabA by multiple STPK. Nine recombinant STPKs (PknA to PknL) encoded by the M. tuberculosis genome were expressed and purified as GST fusions and incubated with purified His-tagged MabA and [γ-³³]ATP. The quantity of the different STPKs varied from 0.6 to 4.2 μg to obtain the optimal autophosphorylation activity for each kinase. Samples were separated by SDS-PAGE and stained with Coomassie Blue (upper panel) and visualized by autoradiography after overnight exposure to a film (lower panel). The upper bands illustrate the autokinase activity of each STPK, and the lower bands represent phosphorylated MabA. B, in vitro phosphorylation of the single and triple MabA mutants by PknB. Four μg of purified MabA_WT, MabA_T21A, MabA_T114A, MabA_T191A, and MabA_T21A/T114A/T191A were incubated individually with PknB and [γ-³³]ATP. Samples were separated by SDS-PAGE and stained with Coomassie Blue (upper panel) and visualized by autoradiography after overnight exposure to a film (lower panel). Upper bands reflect the intense autokinase activity of PknB, and the lower bands illustrate the phosphorylation state of each MabA variant following in vitro phosphorylation by PknB.

FIGURE 3.

Conservation of phosphoacceptors in MabA orthologues. A, multiple sequence alignment of MabA sequences from various mycobacterial species. The alignment was performed using ClustalW and Espript (M_tub, M. tuberculosis; M_bov, M. bovis; M_abs, M. abcessus; M_lep, Mycobacterium leprae; M. avi_para; Mycobacterium paratuberculosis; M_avi, Mycobacterium avium; M_sme, Mycobacterium smegmatis; M_mar, Mycobacterium marinum). Residues conserved in all species are shown in black boxes. The three phosphorylation sites of MabA_Mtb are indicated. Protein secondary element assignments are represented above the sequences. Numbering of amino acids corresponds to the MabA protein from M. tuberculosis. B, localization of the identified phosphorylation sites in the three-dimensional structure of MabA. (1) MabA in complex with NADPH and the acyl-C4 substrate. The model of the ternary complex MabA-cofactor-substrate was obtain after superposition of the crystal structure of MabA with different homologous ternary complexes. The three phosphorylation sites identified by mass spectrometry are represented in red. (2) The same complex has been redrawn with the color representing crystal structure B factor of the MabA holo-form (PDB code 1UZM). The residues with the lower B factor and mobility are represented in blue, whereas those with the higher B factor and mobility are in green and yellow. Only Thr¹⁹¹ lies in a high mobility zone and probably accessible to the kinase. (3) Close-up of the substrate and cofactor binding pocket centered on residue Thr¹⁹¹. The lateral chain of Thr¹⁹¹ is 4.7 Å from the substrate and 3.6 Å from the cofactor. Addition of the phospho group on the oxygen of the lateral chain of Thr¹⁹¹ of MabA is incompatible with substrate binding.

FIGURE 4.

KAR activity of MabA_WT and mutant derivatives. A, reaction scheme for the reduction of acetoacetyl-CoA by MabA. B, MabA_WT, as well as MabA_T191A and MabA_T191D mutants, were purified from recombinant E. coli, dialyzed, and assayed for KAR activity in the presence of acetoacetyl-CoA and NADPH in 100 mm HEPES, pH 7.0, at 25 °C. Conversion of NADPH to NADP was monitored spectrophotometrically at 340 nm. The activity of MabA_WT was arbitrarily fixed at 100%. The values are means of triplicates and are representative of two sets of experiments done with independent protein preparations.

FIGURE 5.

Fluorescence emission of MabA_WT and Thr¹⁹¹ derivatives in the presence of increasing concentrations of NADPH. The fluorescence emission of MabA_WT was compared with those of the MabA_T191A and MabA_T191D mutants. AU, arbitrary unit. Similar data were obtained using two independent protein preparations.

FIGURE 6.

In vivo effects of M. bovis BCG strains overexpressing MabA_T191A and MabA_T191D. A, alteration of mycobacterial growth. Electrocompetent M. bovis BCG 1173P2 cells were transformed with the empty pMK1 construct, the pMK1_mabA_WT, pMK1_mabA_T191A, or pMK1_mabA_T191D to allow constitutive expression of the mabA alleles under control of the strong hsp60 promoter. Transformed mycobacteria were plated and incubated at 37 °C for 3 (for pMK1) or 4 weeks (for pMK1_mabA_WT, pMK1_mabA_T191A, or pMK1_mabA_T191D). B, MabA and InhA expression levels in the M. bovis BCG strains. Western blot analysis of M. bovis BCG cells were transformed with pSD26_mabA_WT, pSD26_mabA_T191A, or pSD26_mabA_T191D and grown in the presence of 50 μg/ml of hygromycin. At an A₆₀₀ of 0.8, expression was induced by adding 0.2% acetamide for 16 h. Bacteria were harvested at various time points, resuspended in PBS, and disrupted. Equal amounts of crude lysates (100 μg) were then loaded onto a 12% acrylamide gel, subjected to electrophoresis, and transferred onto a membrane for immunoblot analysis using either rat anti-MabA antibodies or rabbit anti-InhA antibodies. Endogenous and recombinant MabA proteins are indicated by arrowheads. The results for two individual clones with each construct are presented. C, the inhibitory effect on the incorporation of [1,2-¹⁴C]acetate was assayed using cultures induced for 16 h after addition of acetamide. After a 6-h period of labeling, cells were recovered and hydrolyzed by adding 15% tetrabutylammonium hydroxide at 100 °C overnight. ¹⁴C-Labeled lipids were extracted, resuspended in CH₂Cl₂, and counted in the presence of scintillation liquid. Counts for the strain overexpressing wild-type MabA were arbitrarily fixed at 100%. Bars represent [¹⁴C]acetate incorporation of two individual clones overexpressing either MabA_T191A or MabA_T191D and are expressed as the mean of three independent experiments ± S.D. D, radiolabeled lipids were subjected to TLC using petroleum ether/acetone (95:5, v/v). Spots corresponding to α- and keto-mycolic acids were quantified using a PhosphorImager system for each strain. Bars represent the percentage of α- and keto-mycolic acids of each strain, with respect to the WT, arbitrarily fixed at 100%. The results for two individual clones overexpressing either MabA_T191A or MabA_T191D are presented and expressed as the mean of three independent experiments ± S.D.