Molecular mechanisms of the adaptation of Actinobacillus pleuropneumoniae to the porcine respiratory tract
Ilse D. Jacobsen
Actinobacillus (A.) pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a disease of economic importance occurring worldwide. The disease is characterized by hemorrhagic and necrotizing lung lesions in combination with a fibrinous to fibrous pleuritis. The pathogen’s ability to survive on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated sequesters is of epidemiological importance, as it leads to clinically healthy carrier animals.
In the present study two-dimensional polyacrylamide gel electrophoresis was used in combination with quadrupole time-of-flight mass spectrometry to identify A. pleuropneumoniae proteins upregulated in an ex vivo model upon induction with bronchoalveolar lavage fluid (BALF). Among these proteins, the aspartate ammonia-lyase (aspartase) was further characterized. This enzyme is involved in the production of fumarate, an alternative electron acceptor under anaerobic conditions.
It has been previously demonstrated that deletion of the anaerobic DMSO reductase gene (dmsA), likewise involved in anaerobic respiration, results in attenuation in acute disease. The gene coding for aspartase, aspA was cloned and shown to be present in all A. pleuropneumoniae serotype reference strains. The transcriptional start point was identified downstream of a putative FNR (HlyX) binding motif, and HlyX-dependency of aspartase upregulation under anaerobic conditions was confirmed using an HlyX-mutant. BALF-dependent activation of aspA was confirmed by construction of an isogenic A. pleuropneumoniae mutant carrying a chromosomal aspA::luxAB transcriptional fusion. Two aspA deletion mutants, A. pleuropneumoniae
∆aspA and A. pleuropneumoniae ∆aspA∆dmsA were constructed, both showing reduced growth under anaerobic conditions in vitro. Pigs challenged with either of the two mutants in an aerosol infection model showed a lower lung lesion score compared to the A. pleuropneumoniae wild type (wt) controls. Pigs challenged with
A. pleuropneumoniae ∆aspA∆dmsA had a significantly lower clinical score, and this mutant was rarely reisolated from unaltered lung tissue; in contrast, A. pleuropneumoniae ∆aspA and A. pleuropneumoniae wt were consistently reisolated in high numbers. These results suggest, that enzymes involved in anaerobic respiration are necessary for the pathogen's ability to persist on respiratory tract epithelium and play an important role in A. pleuropneumoniae pathogenesis.
In previous studies BALF was shown to induce expression of putatively Fur-regulated proteins (HENNIG et al. 1999), such as TbpB, which is essential for establishment of A. pleuropneumoniae infection. The alteration of gene expression in response to iron restriction in various bacteria is known to be largely mediated by the ferric uptake regulator Fur. To further investigate the role of Fur in BALF-induced expression of A. pleuropneumoniae proteins, the isogenic in-frame deletion mutant A. pleuropneumoniae ∆fur was constructed. This mutant showed growth deficiencies on PPLO agar plates and in liquid medium under aerobic and anaerobic conditions.
Further, the mutant did not grow on selective agar containing bacitracin; this effect was abolished by addition of an iron chelator to the selective agar, thereby implying that sensitivity to this antibiotic depends on iron availability. Expression of transferrin-binding proteins in A. pleuropneumoniae ∆fur was investigated, and it could be demonstrated, that iron-mediated repression of these proteins depends on Fur. To investigate virulence of A. pleuropneumoniae ∆fur, an aerosol infection experiment was performed. The mutant was found to be significantly attenuated but still able to consistently colonize and cause both clinical disease and lung lesions. This clearly demonstrates the importance of Fur-mediated gene regulation for A. pleuropneumoniae virulence.
In order to identify additional BALF-induced proteins that might belong to the Fur regulon, a differential proteome analysis of surface-associated protein preparations of A. pleuropneumoniae wt grown under standard conditions and after induction with BALF were compared to A. pleuropneumoniae ∆fur. Five of the twelve proteins upregulated by BALF were also found to be upregulated in A. pleuro-pneumoniae ∆fur. Of these five proteins four could be identified by mass spectrometry as the heat shock chaperonine GroES, a binding protein for a putative dipeptide transport system, part of a putative metal ion transport system and an uncharacterized protein conserved in bacteria, respectively. Upstream of the genes
coding for the later two, putative Fur boxes were identified. Iron supplementation did not influence expression of these proteins, suggesting that yet unknown factors in the BALF rather than iron deficiency are responsible for the observed upregulation of Fur-regulated proteins in BALF-induced cultures. Of the remaining proteins induced by BALF, four could be identified; one of these is involved in mRNA stabilization and has been previously reported to be important for A. pleuropneumoniae survival in the host.
In summary, this study showed that differential proteome analysis can be a valuable tool to complement and extend transcriptome studies aimed at unravelling molecular mechanisms of bacterial adaptation to different environments. In combination with an ex vivo model, this approach was successfully used to identify proteins important for A. pleuropneumoniae virulence. Members of both the Fur and the HlyX regulon were found to be upregulated in the ex vivo model in this study. Future work aimed at a comprehensive analysis of both the Fur and the HlyX regulon, possibly by employing proteomic methods to A. pleuropneumoniae ∆fur and A. pleuropneumoniae ∆hlyX mutants, could significantly expand our understanding of the pathways that allow A. pleuropneumoniae to survive and persist in the host.