In Staphylococcus aureus, the acyl-CoA synthetase MbcS supports branched-chain fatty acid synthesis from carboxylic acid and aldehyde precursors

Abstract

In the human pathogen Staphylococcus aureus, branched-chain fatty acids (BCFAs) are the most abundant fatty acids in membrane phospholipids. Strains deficient for BCFAs synthesis experience auxotrophy in laboratory culture and attenuated virulence during infection. Furthermore, the membrane of S. aureus is among the main targets for antibiotic therapy. Therefore, determining the mechanisms involved in BCFAs synthesis is critical to manage S. aureus infections. Here, we report that the overexpression of SAUSA300_2542 (annotated to encode an acyl-CoA synthetase) restores BCFAs synthesis in strains lacking the canonical biosynthetic pathway catalyzed by the branched-chain α-keto acid dehydrogenase (BKDH) complex. We demonstrate that the acyl-CoA synthetase activity of MbcS activates branched-chain carboxylic acids (BCCAs), and is required by S. aureus to utilize the isoleucine derivative 2-methylbutyraldehyde to restore BCFAs synthesis in S. aureus. Based on the ability of some staphylococci to convert branched-chain aldehydes into their respective BCCAs and our findings demonstrating that branched-chain aldehydes are in fact BCFAs precursors, we propose that MbcS promotes the scavenging of exogenous BCCAs and mediates BCFA synthesis via a de novo alternative pathway.

Keywords: Staphylococcus aureus; BKDH complex; MRSA; branched‐chain fatty acids; fatty acids metabolism; membrane phospholipids.

FIGURE 1

Canonical pathway for the synthesis of BCFAs via the BKDH complex. The BCAAs isoleucine (Ile), leucine (Leu), and valine (Val) are converted into their respective α‐keto acids by the transaminase IlvE. α‐KMV (α‐keto‐β‐methylvalerate), α‐KIC (α‐ketoisocaproate), and α‐KIV (α‐ketoisovalerate) undergo oxidative decarboxylation catalyzed by the α‐keto acid dehydrogenase (BKDH) complex. The respective acyl‐CoA primers, once condensed with malonyl‐CoA via 3‐ketoacyl‐ACP synthase III (FabH), are elongated into their respective BCFAs by the type II fatty acid synthase (FASII).

FIGURE 2

The putative acyl‐CoA synthetase gene mbcS contributes to BCFAs synthesis in the absence of an active BKDH complex. Wild‐type (WT), lpdA mutant, lpdA suppressor mutant with a modified mbcS promoter (lpdA mbcS1) and lpdA mbcS double mutant cells were grown in (a) TSB, (b) TSB supplemented with a mixture of 0.5 mM BCCAs iC4, iC5, and aC5 or (c) TSB supplemented with 0.5 mM a15:0 fatty acid, and growth behavior was monitored over time as an increase in optical density at 600 nm (OD₆₀₀). Data are plotted as mean ± SD of three biological replicates. ****p < 0.0001, using two‐way ANOVA with Tukey's multiple comparison test at 5–8 h. In panel a, asterisks indicate that the lpdA single mutant and the lpdA mbcS double mutant are significantly different compared to WT and lpdA mbcS1. In panels b and c, asterisks indicate that the lpdA mbcS double mutant is significantly different compared to WT, the lpdA single mutant, and the lpdA mbcS1 mutant.

FIGURE 3

Mutations in the mbcS promoter increase its activity and result in the synthesis of i14:0 BCFAs. (a) Selected alleles identified during suppressor analysis are compared to the WT allele of the mbcS promoter region. Changes are highlighted in magenta, and −10 and −35 boxes are indicated by the gray boxes. (b) WT cells harboring plasmids with either WT or mutant mbcS promoter regions fused translationally to gfp (P _mbcS ‐gfp) were grown to stationary phase in TSB, at which time cells were pelleted and washed with PBS. Promoter activity was then measured (relative fluorescence units [RFUs]; GFP/OD₆₀₀). ****p < 0.0001, one‐way ANOVA with Tukey's multiple comparison test. (c) Suppressor mutants with the indicated mbcS alleles were grown to exponential phase in TSB. Cells were washed with PBS, and membrane fatty acid content was analyzed by GC‐FAME. Data are presented as mean ± SD from three biological replicates. ***p < 0.001, **p < 0.01, one‐way ANOVA with Tukey's multiple comparison test; ns, not significant.

FIGURE 4

Acyl‐CoA synthetase activity of MbcS restores the growth of a S. aureus lpdA mbcS strain in rich, complex medium lacking BCFAs. (a) Clustal Omega (Sievers et al., 2011) was used to align MbcS with amino acid sequences of previously characterized acyl‐CoA synthetases. Amino acids that constitute a conserved motif in the C‐terminal catalytic domain are shown. Amino acids strictly conserved in the selected proteins are shaded; the conserved lysine residue required for the catalytic activity of acyl‐CoA synthetases is highlighted in yellow. Acs, acetyl‐CoA synthetase from Salmonella enterica (Starai & Escalante‐Semerena, 2004); Acs2, acetyl‐CoA synthetase from Saccharomyces cerevisiae (Starai & Escalante‐Semerena, 2004); AcsA, acetyl‐CoA synthetase from B. subtilis (Gardner et al., 2006); IbuA, isobutyryl‐CoA synthetase from R. palustrus (Crosby et al., ; Crosby & Escalante‐Semerena, 2014); FcsA, fatty acyl‐CoA synthetase from R. palustris (Crosby et al., 2012). (b) WT, mbcS, or lpdA mbcS strains containing either the empty integration vector pCT3 (vector‐only control [VOC]), pCT3 containing the WT allele of S. aureus mbcS under the control of the anhydrotetracycline‐inducible tet promoter (pSambcS ⁺), or the allele of mbcS that codes for a lysine‐to‐alanine substitution at the residue 510 [pSambcS(K510A)] were grown in TSB without anhydrotetracycline (‐aTc) and growth (OD₆₀₀) was monitored over time. (c, d) WT or lpdA mbcS strains with the empty vector control or the wild‐type allele of ibuA from R. palustris (pRpibuA ⁺) (c) without or (d) with 25 ng mL⁻¹ of aTc as gratuitous inducer were grown in TSB and OD₆₀₀ was monitored over time. Data are plotted as mean ± SD from three biological replicates. ****p < 0.0001, two‐way ANOVA with Tukey's multiple comparison test. ns, not significant. In panel b, asterisks indicate that lpdA mbcS + VOC and lpdA mbcS + pSambcS(K510A) are statistically different from WT + VOC, mbcS + VOC and lpdA mbcS + pSambcS ⁺. In panel c, asterisks indicate that lpdA mbcS + VOC and lpdA mbcS + pRpibuA ⁺ are statistically different from WT + VOC. In panel d, asterisks indicate that lpdA mbcS + VOC is statistically different from WT + VOC, and lpdA mbcS + pRpibuA ⁺.

FIGURE 5

MbcS is a methylbutyryl‐CoA synthetase. (a) The activity of MbcS was tested in vitro with several short, straight and branched carboxylic acids. Acids are indicated as C _X , where X denotes the carbon length. Data are plotted as the mean specific activity of the enzyme ± SD of at least three independent trials. (b) SaMbcS was incubated with 2.5 mM MgATP, 493 μM coenzyme A (CoA) and 73 μM isobutyric acid (IB) to produce isobutyryl‐CoA as described in Experimental Procedures. Detection and quantification of CoA and IB‐CoA was measured via LC–MS. HK: heat killed enzyme. LC–MS traces are representative of three independent trials.

FIGURE 6

S. aureus utilizes branched‐chain aldehydes to produce BCFAs in a MbcS‐dependent manner. WT, lpdA mutant, lpdA mbcS1 suppressor mutant, and lpdA mbcS double mutant cells were inoculated into chemically defined medium (CDM) (black) or CDM supplemented with either vehicle (DMSO; gray), iC₅ (2 MB; pink), 2‐methylbutyraldehyde (2MA; orange), or a17:0 fatty acid (green). The cell density (OD₆₀₀) was measured after overnight incubation (16–18 h of growth). Data are plotted as mean ± SD from three biological replicates. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, one‐way ANOVA with Tukey's multiple comparison test for each genotype; ns, not significant.

FIGURE 7

Metabolites from S. epidermidis support growth of S. aureus in an MbcS‐dependent manner. (a) The lpdA mutant (Left) and the lpdA mbcS double mutant (Right) were inoculated ~5 mm apart from S. epidermidis on TSA plates and incubated for 24 h. Arrow head indicates the point of inoculation. (b) WT, lpdA mutant, and lpdA mbcS double mutant cells were inoculated into chemically defined medium (CDM) (black) or CDM supplemented with a mixture of aC5, iC4, and iC5 (gray), a17:0 fatty acid (orange), or 10% (green), 1% (pink) or 0.1% (blue), of conditioned CDM from S. epidermidis (i.e., cell‐free supernatant). The cell density (OD₆₀₀) was measured after overnight incubation (16–18 h of growth). Data are plotted as mean ± SD from three biological replicates. ****p < 0.0001, *p < 0.05, one‐way ANOVA with Tukey's multiple comparison test; ns, not significant.

FIGURE 8

Working model for the synthesis of BCFAs in S. aureus in a BKDH‐independent manner. In a lpdA mutant the BKDH complex is inactive and during laboratory cultivation the synthesis of BCFAs is blocked. S. aureus strains with high MbcS enzyme activity (i.e., the overexpression of mbcS) can synthesize BCFAs independent of the BKDH complex using exogenous or endogenous precursors. We propose α‐keto acids are converted into their respective branched‐chain aldehydes by an α‐keto acid decarboxylase, followed by a reaction catalyzed by an aldehyde dehydrogenase to generate BCCAs. MbcS‐dependent acyl‐CoA synthesis feeds FASII to generate BCFAs for incorporation into membrane phospholipids. Whether the two pathways operate in parallel or function under specific conditions is a focus of ongoing research. BrnQ, BCAA permease; IlvE, BCAA aminotransferase.