Analysis of the Actinobacillus pleuropneumoniae ArcA regulon identifies fumarate reductase as a determinant of virulence

Abstract

The ability of the bacterial pathogen Actinobacillus pleuropneumoniae to grow anaerobically allows the bacterium to persist in the lung. The ArcAB two-component system is crucial for metabolic adaptation in response to anaerobic conditions, and we recently showed that an A. pleuropneumoniae arcA mutant had reduced virulence compared to the wild type (F. F. Buettner, A. Maas, and G.-F. Gerlach, Vet. Microbiol. 127:106-115, 2008). In order to understand the attenuated phenotype, we investigated the ArcA regulon of A. pleuropneumoniae by using a combination of transcriptome (microarray) and proteome (two-dimensional difference gel electrophoresis and subsequent mass spectrometry) analyses. We show that ArcA negatively regulates the expression of many genes, including those encoding enzymes which consume intermediates during fumarate synthesis. Simultaneously, the expression of glycerol-3-phosphate dehydrogenase, a component of the respiratory chain serving as a direct reduction equivalent for fumarate reductase, was upregulated. This result, together with the in silico analysis finding that A. pleuropneumoniae has no oxidative branch of the citric acid cycle, led to the hypothesis that fumarate reductase might be crucial for virulence by providing (i) energy via fumarate respiration and (ii) succinate and other essential metabolic intermediates via the reductive branch of the citric acid cycle. To test this hypothesis, an isogenic A. pleuropneumoniae fumarate reductase deletion mutant was constructed and studied by using a pig aerosol infection model. The mutant was shown to be significantly attenuated, thereby strongly supporting a crucial role for fumarate reductase in the pathogenesis of A. pleuropneumoniae infection.

FIG. 1.

Functional classification of ArcA-regulated genes according to the COGs database. The classification was adopted from the genome annotation for A. pleuropneumoniae serotype 5b L20 from the NCBI database (GenBank accession no. CP000569).

FIG. 2.

2D DIGE analysis. The A. pleuropneumoniae wt (shown here labeled with Cy5 [red]) and A. pleuropneumoniae ΔarcA (shown here labeled with Cy3 [green]) proteins were precipitated directly from whole-cell lysates and separated by using Immobiline DryStrips with pI gradients of 4 to 7 (A) or 7 to 11 (B). The A. pleuropneumoniae wt and A. pleuropneumoniae ΔarcA mutant were grown anaerobically in four independent cultures (each). By using three fluorescent dyes, Cy2 (blue [internal standard]), Cy3 (green [either wt or ΔarcA]) and Cy5 (red [either wt or ΔarcA]), on each gel, an internal standard and a protein preparation of the A. pleuropneumoniae wt and A. pleuropneumoniae ΔarcA mutant were separated simultaneously. Fluorescence was detected using a Typhoon Trio fluorescence scanner. Quantitative and statistical analyses of differences in protein expression levels were performed with DeCyder 2D 6.5 software. Each gel shown here represents one out of four gels that were run and analyzed. Arrowheads highlight protein spots that were significantly (P ≤ 0.05) upregulated in the presence of ArcA. Arrows indicate protein spots that were significantly (P ≤ 0.05) downregulated in the presence of ArcA. Protein spots of interest were excised from preparative gels, trypsinized and, after recovery of peptides from the gel, analyzed by MS.

FIG. 3.

Impact of ArcA-regulated genes on metabolic pathways. (A) Glycolysis and citric acid cycle. (B) Dehydrogenases, terminal reductases, and membrane transporter. (C) Fermentation. (A to C) T, transcript; P, protein. The degree of ArcA-dependent regulation is given within the boxes when results obtained by microarray analysis (transcript) or by 2D DIGE (protein) were statistically significant (P ≤ 0.05; gray-highlighted box, upregulated in the presence of ArcA; unhighlighted box, downregulated in the presence of ArcA). Dehydrogenases are indicated by a rounded rectangle, terminal reductases are indicated by hexagons, and membrane transporters are indicated by rounded rectangle pairs. Biochemical pathways were adopted from those for E. coli K-12 (BioCyc, http://www.biocyc.org). The sequence and annotation of the A. pleuropneumoniae serotype 5b strain L20 genome have recently been published (GenBank accession no. CP000569). If A. pleuropneumoniae carries no homologue to the respective E. coli protein, its name is crossed out.

FIG. 3.

Impact of ArcA-regulated genes on metabolic pathways. (A) Glycolysis and citric acid cycle. (B) Dehydrogenases, terminal reductases, and membrane transporter. (C) Fermentation. (A to C) T, transcript; P, protein. The degree of ArcA-dependent regulation is given within the boxes when results obtained by microarray analysis (transcript) or by 2D DIGE (protein) were statistically significant (P ≤ 0.05; gray-highlighted box, upregulated in the presence of ArcA; unhighlighted box, downregulated in the presence of ArcA). Dehydrogenases are indicated by a rounded rectangle, terminal reductases are indicated by hexagons, and membrane transporters are indicated by rounded rectangle pairs. Biochemical pathways were adopted from those for E. coli K-12 (BioCyc, http://www.biocyc.org). The sequence and annotation of the A. pleuropneumoniae serotype 5b strain L20 genome have recently been published (GenBank accession no. CP000569). If A. pleuropneumoniae carries no homologue to the respective E. coli protein, its name is crossed out.

FIG. 3.

Impact of ArcA-regulated genes on metabolic pathways. (A) Glycolysis and citric acid cycle. (B) Dehydrogenases, terminal reductases, and membrane transporter. (C) Fermentation. (A to C) T, transcript; P, protein. The degree of ArcA-dependent regulation is given within the boxes when results obtained by microarray analysis (transcript) or by 2D DIGE (protein) were statistically significant (P ≤ 0.05; gray-highlighted box, upregulated in the presence of ArcA; unhighlighted box, downregulated in the presence of ArcA). Dehydrogenases are indicated by a rounded rectangle, terminal reductases are indicated by hexagons, and membrane transporters are indicated by rounded rectangle pairs. Biochemical pathways were adopted from those for E. coli K-12 (BioCyc, http://www.biocyc.org). The sequence and annotation of the A. pleuropneumoniae serotype 5b strain L20 genome have recently been published (GenBank accession no. CP000569). If A. pleuropneumoniae carries no homologue to the respective E. coli protein, its name is crossed out.

FIG. 4.

Histopathological examination of lung lesions. Sections were obtained from pigs necropsied on day 7 postinfection or on day 21 postinfection. Animals were infected with either the A. pleuropneumoniae wt or the A. pleuropneumoniae Δfrd mutant. A, alveole; B, bronchus; F, fibrin; IC, interstitial consolidation.