Identification of dimethyl sulfoxide reductase in Actinobacillus pleuropneumoniae and its role in infection

Abstract

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is capable of persisting in oxygen-deprived surroundings, namely, tonsils and sequestered necrotic lung tissue. Utilization of alternative terminal electron acceptors in the absence of oxygen is a common strategy in bacteria under anaerobic growth conditions. In an experiment aimed at identification of genes expressed in vivo, the putative catalytic subunit DmsA of anaerobic dimethyl sulfoxide reductase was identified in an A. pleuropneumoniae serotype 7 strain. The 90-kDa protein exhibits 85% identity to the putative DmsA protein of Haemophilus influenzae, and its expression was found to be upregulated under anaerobic conditions. Analysis of the unfinished A. pleuropneumoniae genome sequence revealed putative open reading frames (ORFs) encoding DmsB and DmsC proteins situated downstream of the dmsA ORF. In order to investigate the role of the A. pleuropneumoniae DmsA protein in virulence, an isogenic deletion mutant, A. pleuropneumoniae DeltadmsA, was constructed and examined in an aerosol infection model. A. pleuropneumoniae DeltadmsA was attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly to A. pleuropneumoniae virulence.

FIG. 1.

PrettyPlot alignment of amino-terminal ends of DmsA proteins from A. pleuropneumoniae, H. influenzae, and E. coli. The TAT pathway consensus sequence T-R-R-X-F-L-K is indicated by boldface italic type, and the putative signal peptidase cleavage site for A. pleuropneumoniae DmsA is indicated by an arrowhead. The PrettyPlot presentation of the amino-terminal end of the mature DmsA protein shows the high degree of homology typical for the entire protein.

FIG. 2.

Expression of the A. pleuropneumoniae DmsA protein as assessed by Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (top panels) and corresponding Western blotting (bottom panels). (A) Expression upon growth under standard conditions (lane 1) and anaerobic conditions (lane 2). (B) Effects of different iron sources and BALF upon growth under aerobic conditions. Lane 1, standard conditions; lane 2, iron restriction; lane 3, addition of BALF; lanes 4 and 5, addition of ferric citrate at final concentrations of 20 and 50 μM, respectively; lanes 6 and 7, addition of ferrous citrate at final concentrations of 20 and 50 μM, respectively.

FIG. 3.

Characterization of the A. pleuropneumoniae dmsA deletion mutant with A. pleuropneumoniae AP76 (lanes 1) and A. pleuropneumoniae ΔdmsA (lanes 2). (A) PCR performed with primers oDMSAdel1 and oDMSAdel2. Lane − contained no DNA template. (B) Southern blot analysis of genomic DNA digested with EcoRI and BspMI, performed with the oDMSAdel1-oDMSAdel2 PCR product as the radiolabeled probe. A BspMI restriction site is located in the deleted fragment. (C) Coomassie blue-stained gel (left) and Western blot (right) developed with polyclonal rabbit antiserum. (D) PFGE analysis, showing that no gross genomic rearrangements occurred. The arrowheads in lanes 1 (parent strain) indicate the fragments that hybridized with a dmsA-derived probe.

FIG. 4.

Virulence of A. pleuropneumoniae ΔdmsA in an aerosol infection model. •, A. pleuropneumoniae wild-type strain AP76 (AP76wt); ▴, A. pleuropneumoniae ΔdmsA (AP76 ΔdmsA). The central symbol in each hourglass shape indicates the geometric mean, the hinges indicate the values in the middle half of the data, and the top and bottom symbols indicate the maximum and minimum values. The asterisks indicate statistical significance (P < 0.05) as determined by the Wilcoxon signed-rank test. (A) Body temperatures of pigs over the course of 8 days. Day 0 was the day of infection. (B) Humoral immune responses of pigs challenged with the A. pleuropneumoniae parent strain and the isogenic mutant 7 days before and 7 and 21 days after challenge. The antibody response was assessed with two ELISAs by using the recombinant ApxIIA protein (Apx ELISA) (open symbols) or a detergent extract (extract ELISA) (solid symbols) as the solid-phase antigen. The immune response was expressed in ELISA units (based on an external standard) for the standardized Apx ELISA, and serum activities of more than 30 ELISA units were considered positive (28); for the extract ELISA, the immune response was expressed as the serum titer compared to the data for an internal control (18). (C) Endoscopic scores 7 days before and 7 and 21 days after challenge.