c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation

Abstract

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the β-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.

Figure 1. Growth of c-di-AMP-deficient mutants in rich medium and resistance to cefuroxime

(A and B) Mutants constructed and grown overnight in LSM were serially diluted 10-fold in a 96-well plate with PBS then 5 μL of each dilution was spotted onto either LSM or BHI agar. Images were taken and CFU were enumerated after 48 hrs of incubation at 37 °C. (C) Immunoblot of DacA and P60 (loading control) proteins for the strains indicated, grown to mid-log in LSM at 37°C. Data are representative of three independent experiments. (D) Antibiotic sensitivity measured by disk diffusion of 125 μg of cefuroxime on LSM-agar for the indicated L. monocytogenes strains measured at 48 hrs. (B and D) Data are mean ± standard error of the mean (s.e.m) of at least three independent experiments.

Figure 2. ΔdacA suppressor mutants

(A and B) Illustration of oligopeptide permease (Opp) operon and protein subunits. (C and D) Illustration of glycine-betaine importer (Gbu) operon and protein subunits. (E) Operon of c-di-AMP binding protein B (cbpB) which encodes a c-di-AMP binding, CBS domain containing, protein of unknown function. ccpC, catabolite control protein and LysR family transcriptional regulator (Kim et al., 2005); dapH, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-acetyltransferase; dapL, N-acetyl-diaminopimelate deacetylase (similar to dapL in B. subtilis). (F) Operon of PII-like signal transduction protein A (pstA) a c-di-AMP binding, PII-like, protein of unknown function. lmo2694, hypothetical ornithine/lysine/arginine decarboxylase (similar to yaaO in B. subtilis); tmk, thymidylate kinase. (A, C, E, and F) Mutations previously identified (Whiteley et al., 2015) and not drawn to scale. (G) Enumeration of CFU on indicated media for L. monocytogenes strains constructed in LSM. “+” vs “Δ” indicate a mutation in a wild-type or dacA-deficient background. Data are mean ± s.e.m of at least three independent experiments.

Figure 3. Growth of ΔdacA in media containing oligopeptides and salt

(A-D) Enumeration of CFU on indicated media for L. monocytogenes strains constructed in LSM. Dashed line indicates limit of detection (Lod). (B) “KLLLLK”, “KAAAAK”, and “AQ” are sequences of synthetic oligopeptides added, and “A&Q” is the molar equivalent of free amino acids (E) Cefuroxime disk diffusion on LSM-agar supplemented with the indicated concentration of NaCl. All data are mean ± s.e.m of at least three independent experiments.

Figure 4. ΔdacA suppressor mutant resistance to cefuroxime

Cefuroxime disk diffusion on LSM-agar of L. monocytogenes strains. Data are mean ± s.e.m of at least three independent experiments.

Figure 5. Suppressor mutations of ΔdacA cefuroxime sensitivity

(A) Illustration of suppressor analysis. (B) Schematic of central metabolism in L. monocytogenes. Grey arrows with red X indicate enzymes not encoded in the L. monocytogenes genome, bold labels indicate enzyme names, and non-bold labels indicate metabolites. Underlined labels indicate metabolic pathways providing or using precursors/products of the enzymes shown. OAA, oxaloacetate; CIT, citrate; ICI, isocitrate; αKG, 2-oxoketoglutarate; SUC, succinate; CoA, coenzyme A; FUM, fumarate; MAL, malate. (C) PycA suppressor mutations from Table 1 on color-coded protein domains showing the biotin carboxylase (BC), pyruvate carboxylase tetramerization (PT), carboxyltransferase (CT), and biotin carboxyl carrier protein (BCCP) domains. Figure not drawn to scale. (D) Crystal structure of PycA (PDB: 4QSH, (Sureka et al., 2014)) from L. monocytogenes with modeled suppressor mutations on all four monomers. Monomer 1 is colored as in (C) and only mutations on this monomer are labeled. The resolved c-di-AMP (cdA) molecules are shown in red. (E) Crystal structure of PycA homolog from S. aureus (PDB: 3HO8, (Yu et al., 2009)) with modeled suppressor mutations at homologous residues on all four monomers. Monomer 1 is colored as in (C) and only mutations near the acetyl-CoA binding site are labeled on this monomer. The resolved coenzyme A ligands are also shown. (F) Detailed view of (E) modeling interactions between the S. aureus arginine residues homologous to R367 and R1051 with coenzyme A. Dashed red lines hydrogen bonds. (D, E, and F) Visualizations made with PyMol software. (G) Enzymatic activity of recombinant PycA in the absence or presence of the allosteric activator acetyl-CoA. Data are the mean ± s.e.m. of three independent experiments and p values were calculated using a heteroscedastic Student's t-test; ** p < 0.001, ns p > 0.05.

Figure 6. ΔdacA suppressor mutations in PycA

(A) Immunoblot of PycA (biotin cofactor using streptavidin) and P60 (loading control). Data are representative of three independent experiments. (B) Enumeration of CFU on indicated media for L. monocytogenes strains constructed in LSM. “+” vs “Δ” indicate a mutation in a wild-type or dacA-deficient background. (C) Cefuroxime disk diffusion on LSM-agar of L. monocytogenes strains. (B and C) Data are the mean ± s.e.m. of at least three independent experiments. (D and E) CFU recovered from spleens 48 hours post-infection of CD-1 mice infected with 10⁵ CFU of the indicated strains. Data are pooled results from two independent experiments of n = 5 mice, black bars indicate median, p values were calculated using a heteroscedastic Student's t-test; ** p < 0.001, ns p > 0.05.

Figure 7. TCA cycle intermediates restrict growth in the absence of c-di-AMP

(A) Enumeration of CFU on indicated media for L. monocytogenes strains constructed in LSM. (B) Cefuroxime disk diffusion on LSM-agar of L. monocytogenes strains. dacA vs. ΔdacA and “+” vs “Δ” indicate a mutation in a wild-type or dacA-deficient background. (C) Intracellular citrate levels normalized to wild-type L. monocytogenes, p values were calculated using a heteroscedastic Student's t-test; * p < 0.05. All data are mean ± s.e.m of at least three independent experiments.