Pleiotropic effect of AccD5 and AccE5 depletion in acyl-coenzyme A carboxylase activity and in lipid biosynthesis in mycobacteria

Abstract

Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs.

Figure 1. Allelic exchange of the accD5 locus of M. smegmatis.

A-C. Genetic organization and partial restriction maps of the accD5 chromosomal region of (A) the wild-type strain mc²155, (B) the single crossover strain D5SCO6, and (C) the M. smegmatis D5DCO conditional mutant. D) Southern blot analysis of M. smegmatis accD5 mutants. Three conditional mutants were picked at random (lanes 1–3), and their chromosomal DNA digested with EcoRI and probed for hybridization with a 390 bp labeled fragment corresponding to the 5′ region of accD5. M. smegmatis mc²155 DNA was included as a control (WT). Molecular masses are indicated in kilobases.

Figure 2. Complementation of the accD5-accE5 conditional mutant.

Schematic representation of the pMP395 derived constructions used in the complementation studies.

Figure 3. accD5-accE5 conditional mutant D5 MUT.

A) Schematic representation of the genetic organization of the M. smegmatis conditional mutant D5 MUT. In pBB25, transcription of the accD5-accE5 operon is controlled by P_tr, which can be repressed with the addition of ATc to the media. B) M. smegmatis mc²155 (WT), Isogenic (ISO-D5) and D5 MUT strains were grown on plates with 200 ng ml⁻¹ (+ATc) or without ATc (−ATc). C) Growth curves of D5 MUT in the presence or absence of ATc 200 ng ml⁻¹. A saturated culture of D5 MUT grown at 37°C was diluted in fresh 7H9 medium to an OD_{600 nm} of 0.01 and further incubated at 37°C. After 5, 8, 11 and 21 h; an aliquot of the main culture was separated and supplemented with ATc 200 ng ml⁻¹. Growth was followed by measuring OD_{600 nm}.

Figure 4. Effect of accD5-E5 expression on D5 MUT growth and cell viability.

A saturated culture of D5 MUT grown at 37°C was diluted in fresh 7H9 medium to an OD_{600 nm} of 0.01 and 9–10 h later (OD_{600 nm}∼0,06) ATc 200 ng ml⁻¹ was added to an aliquot of the culture. A) Growth was followed by measuring OD_{600 nm}. Arrows indicate the times when aliquots of the cultures were collected for further analysis (T1, T2, T3 and T4). B) The number of viable cells of D5 MUT in the cultures grown in presence or absence of ATc was evaluated by plating serial dilutions onto LB plates at 37°C. C) Western blot analysis of total crude lysates from D5 MUT strain grown with (+) and without (−) ATc 200 ng ml⁻¹. Detection was performed using anti-AccD5 antibodies elicited in rabbit (upper panel) and anti-KasA as loading control (lower panel).

Figure 5. Acyl-CoA carboxylase activity in D5 MUT.

Cell-free extracts were prepared from D5 MUT incubated in absence and presence of ATc 200 ng ml⁻¹ at the times indicated in Fig. 4A. Determination of A) Acetyl-CoA carboxylase (ACC), B) Propionyl-CoA carboxylase (PCC), C) Long-chain acyl-CoA carboxylase and D) Malic enzyme activities. Levels of activity are the means of the results of three independent experiments ± standard deviations (n = 3). ***, P = 0.0001; *, P = 0.04.

Figure 6. Acyl-CoA pool in D5 MUT.

Acyl-CoAs were extracted from the same number of cells of cultures incubated in the absence or presence of ATc during ∼10 h (T2). Samples were analyzed by LC-MS. A) Short chain acyl-CoAs. B) Medium and long chain acyl-CoAs. C) Very long chain acyl-CoAs. Results are the means of three independent experiments ± standard deviations (n = 3). *, P = 0.04. cC24: carboxy-C₂₄-CoA.

Figure 7. Fatty and mycolic acid profiles in D5 MUT.

A) de novo fatty acid and mycolic acid biosyntheses. At the indicated times, aliquots from D5 MUT cultures incubated in absence or presence of ATc were labelled with [¹⁴C]-acetate for 1 hour at 37°C. Fatty acid and mycolic acids methyl esters were extracted from samples containing equivalent amounts of bacteria and were analyzed by TLC. B) Quantification of the radiolabelling intensity of the TLC showed in panel A.