Identification and preliminary characterization of a 75-kDa hemin- and hemoglobin-binding outer membrane protein of Actinobacillus pleuropneumoniae serotype 1

Abstract

The reference strains representing serotypes 1 to 12 of Actinobacillus pleuropneumoniae biotype 1 were examined for their ability to utilize porcine hemoglobin (Hb) or porcine hemin (Hm) as iron sources for growth. In a growth promotion assay, all of the reference strains were able to use porcine Hb, and all strains except 2 were able to use porcine Hm. Using a preliminary characterization procedure with Hm- or Hb-agarose, Hm- and Hb-binding outer membrane proteins (OMPs) of approximately 75 kDa were isolated from A. pleuropneumoniae serotype 1 strain 4074 grown under iron-restricted conditions. Matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) analysis revealed a number of common tryptic peptides between the Hb-agarose- and Hm-agarose-purified 75 kDa OMPs, strongly suggesting that these peptides originate from the same protein. A database search of these peptide sequences revealed identities with proteins from various Gram-negative bacteria, including iron-regulated OMPs, transporter proteins, as well as TonB-dependent receptors. Taken together, our data suggest that A. pleuropneumoniae synthesizes potential Hm- and Hb-binding proteins that could be implicated in the iron uptake from porcine Hb and Hm.

Figure 1. (A) Identification of Hb-binding outer membrane proteins (OMP) of Actinobacillus pleuropneumoniae strain 4074 using affinity chromatography. A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel stained with Coomassie blue is shown. Molecular mass protein markers in kDa (lane 1); A. pleuropneumoniae grown under iron-sufficient conditions (lane 2); OMPs from A. pleuropneumoniae grown under iron-restricted conditions (lane 3) or grown under iron-sufficient conditions (lane 4); hemoglobin (Hb)-agarose control (lane 5); Hb-agarose affinity-purified OMPs from A. pleuropneumoniae grown under iron-restricted conditions (lane 6); Hb-agarose affinity-purified OMPs from A. pleuropneumoniae grown under iron-sufficient conditions (lane 7); Arrows indicate the position of the 75- and 104-kDa Hb-binding OMPs.

Figure 2. Fluorographic analysis of Actinobacillus pleuropneumoniae strain 4074 metabolically labelled with [³H]palmitic acid. Molecular mass protein markers in kDa (lane 1); A. pleuropneumoniae grown under iron-sufficient conditions (lane 2) or iron-restricted conditions (lane 3).

Figure 3. Mass spectral analysis of tryptic peptides of the Actinobacillus pleuropneumoniae 75-kDa outer membrane proteins (OMP) purified by either hemin (Hm)- or hemoglobin (Hb)-agarose. Nanoelectrospray mass spectral analysis of HPLC fraction 11. Conventional mass spectrum (A) showing 3 abundant doubly-protonated ions at m/z 540.7 (1077.4 Da), 662.2 (1322.4 Da), and 736.8 (1471.6 Da) and tandem mass spectrum of m/z 662.2 (1322.4 Da), (B) indicating a series of y-type fragment ions consistent with consecutive cleavage of the peptide bonds. Spacing between adjacent fragment ions allowed to deduce the peptide sequence YDYYDLDNDK.