Link between Heterotrophic Carbon Fixation and Virulence in the Porcine Lung Pathogen Actinobacillus pleuropneumoniae

Abstract

Actinobacillus pleuropneumoniae is a capnophilic pathogen of the porcine respiratory tract lacking enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle. We previously claimed that A. pleuropneumoniae instead uses the reductive branch in order to generate energy and metabolites. Here, we show that bicarbonate and oxaloacetate supported anaerobic growth of A. pleuropneumoniae Isotope mass spectrometry revealed heterotrophic fixation of carbon from stable isotope-labeled bicarbonate by A. pleuropneumoniae, which was confirmed by nano-scale secondary ion mass spectrometry at a single-cell level. By gas chromatography-combustion-isotope ratio mass spectrometry we could further show that the labeled carbon atom is mainly incorporated into the amino acids aspartate and lysine, which are derived from the TCA metabolite oxaloacetate. We therefore suggest that carbon fixation occurs at the interface of glycolysis and the reductive branch of the TCA cycle. The heme precursor δ-aminolevulinic acid supported growth of A. pleuropneumoniae, similar to bicarbonate, implying that anaplerotic carbon fixation is needed for heme synthesis. However, deletion of potential carbon-fixing enzymes, including PEP-carboxylase (PEPC), PEP-carboxykinase (PEPCK), malic enzyme, and oxaloacetate decarboxylase, as well as various combinations thereof, did not affect carbon fixation. Interestingly, generation of a deletion mutant lacking all four enzymes was not possible, suggesting that carbon fixation in A. pleuropneumoniae is an essential metabolic pathway controlled by a redundant set of enzymes. A double deletion mutant lacking PEPC and PEPCK was not impaired in carbon fixation in vitro but showed reduction of virulence in a pig infection model.

Keywords: Actinobacillus pleuropneumoniae; carbon fixation; heme; oxaloacetate; phosphoenolpyruvate; swine; virulence.

FIG 1

Growth of A. pleuropneumoniae in autoclaved PPLO medium. (A) Determination of the concentration-dependent effect of NaHCO₃ on growth of A. pleuropneumoniae. Bacteria were grown in autoclaved PPLO medium supplemented with glucose, NAD, and various concentrations of NaHCO₃ in closed tubes without shaking at 37°C. Bars represent mean OD₆₀₀ values with standard deviations (n = 5) upon 6 h of growth. Three asterisks indicate a P value by Student's t test below 0.001. (B) Semilogarithmic presentation of growth curves of A. pleuropneumoniae wild-type (WT), ΔpepC, ΔpckA, ΔmaeB, ΔoadA, ΔpepC ΔpckA, and ΔpepC ΔpckA ΔmaeB strains upon supplementation of autoclaved PPLO medium (containing glucose and NAD) with 5 mM NaHCO₃, 2 mM oxaloacetic acid (OxHAc), or 5 mM sodium chloride for the indicated time. Symbols represent the mean OD₆₀₀ values at the indicated time points postinoculation. For individual growth curves and number of replicates, see Fig. S3 in the supplemental material. (C) Growth of A. pleuropneumoniae wild-type (WT), ΔpepC ΔpckA, ΔpepC ΔpckA ΔmaeB, and ΔoadA ΔmaeB ΔpckA strains in autoclaved and additionally degassed PPLO medium (containing glucose and NAD) upon supplementation with either 5 mM NaHCO₃, 2 mM oxaloacetic acid, 2 mM aspartic acid, 2 mM malic acid, 2 mM sodium fumarate, 2 mM δ-aminolevulinic acid (ALA), or a solution containing 30 μM heme and 129 μM l-histidine. Bars represent mean OD₆₀₀ values and standard deviations (n = 3) upon 6 h of growth. Statistics were performed by Student's t test comparing each supplemented sample to the negative-control (w/o) for each strain. P values below 0.05, 0.01, and 0.001 are indicated by one, two, or three asterisks, respectively.

FIG 2

Fixation of carbon from NaHCO₃ by A. pleuropneumoniae. (A) NanoSIMS analysis of A. pleuropneumoniae grown for 6 h in the presence of either NaH¹²CO₃ or NaH¹³CO₃. Shown are secondary electron images (SE), ¹²C¹⁴N/¹²C normalized images, and ¹³C/¹²C isotope ratio images. (B) Upon nanoSIMS analysis, 25 and 37 cells grown in the presence of NaN¹²CO₃ or NaH¹³CO₃, respectively, were randomly selected, and their atom% ¹³C values were plotted. The mean values are depicted by the gray lines. Three asterisks indicate a P value by Student's t test below 0.001. (C) ¹³C/¹²C isotope ratios in total biomass of A. pleuropneumoniae wild-type (WT) (n = 4), ΔpepC (n = 4), ΔpckA (n = 4), ΔmaeB (n = 5), ΔoadA (n = 1), ΔpepC ΔpckA (n = 4), and ΔpepC ΔpckA ΔmaeB (n = 3) strains upon growth for 6 h in the presence of NaH¹²CO₃ or NaH¹³CO₃. Error bars represent standard deviations from at least three independent biological repeats.

FIG 3

Incorporation of carbon from NaHCO₃ into amino acids by A. pleuropneumoniae. (A) Analysis of ¹³C/¹²C incorporation ratios for different amino acids at certain time points during growth of A. pleuropneumoniae wild type (WT) in medium containing NaH¹³CO₃. (B) Mathematical fitting of incorporation ratios measured for Asp and Lys using the Chapman-Richards equation, Δδ¹³C = a(1 − e^(−bt))^c. Red circles and green triangles represent measured values, and red and green crosses that are connected by a line represent values calculated with the Chapman-Richards equation for Asp and Lys, respectively. In panels A and B, incorporation ratios for Asp refer to the primary (left) y axis, and all other amino acids refer to the secondary (right) y axis.

FIG 4

Analysis of A. pleuropneumoniae ΔpepC ΔpckA strain virulence using aerosol infection of pigs. (A) Time course of body temperature of animals infected with A. pleuropneumoniae wild-type or ΔpepC ΔpckA strain. Animals were infected on day 0. Body temperature was recorded starting 4 days before infection and during the following 6 days after experimental infection. Depicted are least-squares (LS) means of body temperatures within each group. A mixed-model repeated-measure analysis with group-day interaction revealed that the interaction between group and day is significant (P = 0.0223). Post hoc analysis was carried out regarding the combinations of fixed factors of interest, and significant differences on individual days are indicated by asterisks (P < 0.05 by Student's t test). Num DF, numerator degrees of freedom; Den DF, denominator degrees of freedom; F value, F value (test statistic); Pr > F, P value. (B) Clinical score, lung lesion score, and reisolation score (as indicated on the y axis) of pigs infected with A. pleuropneumoniae wild-type or ΔpepC ΔpckA strain. Each symbol represents one animal (triangles, two animals of the wild-type-infected group that had to be sacrificed within 3 days after infection because of animal welfare reasons; circles, six animals of the wild-type group that were sacrificed on day 21; rectangles, eight animals of the ΔpepC ΔpckA strain-infected group that were sacrificed on day 21). The horizontal lines represent the arithmetic means. Differences between A. pleuropneumoniae wild-type and ΔpepC ΔpckA strains were statistically analyzed using the Mann-Whitney U test and P values are depicted.

FIG 5

Scheme of glycolysis and reductive TCA cycle. Metabolites of glycolysis and TCA cycle and their contribution as precursors for amino acids are shown according to BioCyc for E. coli K-12 substrain MG1655 (www.biocyc.org). Metabolic pathways were deduced from the enzymatic repertoire of A. pleuropneumoniae, which is derived from the homology-based annotation of the A. pleuropneumoniae genome. Potential direct enzymatic interconversions are depicted by black arrows, and reactions including several steps are shown by gray arrows. Double arrows indicate reactions that are potentially reversible. Enzymatic reactions that are probably missing in A. pleuropneumoniae are shown by dashed lines. Amino acids that could be analyzed by GC-C-IRMS are highlighted by boxes with solid lines. Depending on the degree of ¹³C enrichments in amino acids, we applied the following color key: Δδ¹³C [‰] ≈ 5,000, red boxes; Δδ¹³C [‰] ≈ 250, orange boxes; Δδ¹³C [‰] ≈ 10, green boxes; no enrichment, blue boxes. Amino acids that were not accessible with the analytical setup are shown in boxes with dashed lines. Potential carboxylation reactions by PEPC, PEPCK, MAE, and OAD are highlighted by boldface arrows. Abbreviations: G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6BP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GADP, glyceraldehyde 3-phosphate; 1,3BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Ac-CoA, acetyl-coenzyme A (CoA); CIT, citrate; ICT, isocitrate; α-KG, α-ketoglutarate; SUC-CoA, succinyl-CoA; SUC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate; Tyr, tyrosine; Phe, phenylalanine; Trp, tryptophan; Ser, serine; Gly, glycine; Cys, cysteine; Ala, alanine; Leu, leucine; Val, valine; Arg, arginine; Glu, glutamic aicd; Gln, glutamine; Pro, proline; Asp, aspartic acid; Asn, asparagine; Met, methionine; Lys, lysine; Thr, threonine; Ile, isoleucine; PEPC, phosphoenolpyruvate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; OAD, oxaloacetate decarboxylase; MAE, malic enzyme.