The role of iron in Actinobacillus pleuropneumoniae infection
Actinobacillus (A.) pleuropneumoniae is an obligatory porcine pathogen that causes a fibrinous and necrotizing pleuropneumonia of significant economic importance. To date, there are no vaccines available that provide satisfactory cross-serovar protection and, in addition, allow differentiation between infected and vaccinated animals. Thus, a live attenuated marker vaccine would be desirable. The ability to acquire essential nutrients in the host is closely related to bacterial virulence. A. pleuropneumoniae possesses high-affinity receptors for porcine transferrin which are functionally dependent on a transmembrane energy coupling mechanism (TonB-ExbBD) for the uptake of iron from transferrin. In this study, components involved in transferrin-bound iron uptake were investigated with regard to their contribution to A. pleuropneumoniae virulence, and representational difference analysis of cDNA was used to identify other virulence-associated genes. An isogenic exbB deletion mutant of A. pleuropneumoniae that was unable to utilize porcine transferrin as the sole iron source in vitro was examined in an aerosol challenge trial. This strain was completely avirulent, which made the construction of isogenic mutants lacking expression of either TbpB, TbpA or both transferrin-binding proteins necessary. In a challenge trial, all mutants were avirulent, thus emphasizing the crucial role of transferrin bound iron uptake in infection. Furthermore, this result implies that the avirulence of the exbB deletion mutant is due to its inability to utilize transferrin-bound iron rather than due to other cell functions requiring the ExbB protein. Using an ex vivo model (addition of bronchoalveolar lavage fluid [BALF] to culture medium) to induce genes normally expressed in the host only, representational difference analysis was performed on A. pleuropneumoniae cDNA. Ten genes present only in cDNA from A. pleuropneumoniae grown BALF were identified. Among them, the genes for a novel ferric compound receptor unique to serotypes 4 and 7 (fhuA) and the catalytic subunit of dimethylsulfoxide reductase (dmsA) were further characterized, and isogenic mutants were constructed. A challenge experiment showed attenuation in acute disease for both strains. Both genes may be useful in the construction of a novel live vaccine strain carrying multiple deletions.