Differential expression of non-cytoplasmic Actinobacillus pleuropneumoniae proteins induced by addition of bronchoalveolar lavage fluid

Actinobacillus (A.) pleuropneumoniae is the causative agent of a porcine pleuropneumonia occurring worldwide. In order to identify novel non-cytoplasmic putative virulence-associated proteins, we prepared fractions enriched in surface-associated proteins for differential proteome analysis by two-dimensional (2D) gel electrophoresis and quadrupole time-of-flight mass spectrometry (Q-Tof MS). Bacteria grown under standard culture conditions were compared to an ex vivo model based on the addition of bronchoalveolar lavage fluid (BALF) to the culture media. Twelve proteins were found to be upregulated upon induction with BALF, among them a superoxide dismutase, a parvulin-like peptidy-prolyl isomerase, a polynucleotide phosphorylase and the highly immunogenic lipoprotein OmlA. Four of the proteins upregulated by BALF were additionally constitutively expressed by an isogenic A. pleuropneumoniae fur deletion mutant and could be identified by Q-Tof MS as the heat shock protein GroES, a putative dipeptide transporter, a putative metal ion transporter and a conserved protein of unknown function. In silico analysis of the putative promoter regions of the encoding genes revealed putative Fur boxes upstream of two genes, one of which encodes part of a putative metal ion transporter. An isogenic mutant with a deletion in this protein was constructed and designated as A. pleuropneumoniae Deltafui. Analysis of the mutant in an aerosol infection model revealed symptoms indistinguishable from those seen upon infection with wild type A. pleuropneumoniae. This result implies that not all proteins upregulated by BALF are directly involved in A. pleuropneumoniae virulence.