No abstract

Actinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrinbinding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenic A. pleuropneumoniae single mutants (⌬exbB and ⌬ureC) and a newly constructed A. pleuropneumoniae double (⌬ureC ⌬exbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniae ⌬exbB mutant nor the double ⌬ureC ⌬exbB mutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence of A. pleuropneumoniae in the porcine respiratory tract. The A. pleuropneumoniae ⌬ureC mutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection the A. pleuropneumoniae ⌬ureC mutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response-as assessed by fluorescenceactivated cell sorter and enzyme-linked immunosorbent spot analyses-revealed a significantly higher number of A. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniae ⌬ureC mutant than in the BALF of those infected with the parent strain. These results imply that A. pleuropneumoniae urease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniae parent strain.Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia, a highly infectious disease of fattening pigs occurring worldwide (12). A number of putative virulence factors, such as Apx toxins, capsule, lipopolysaccharide (LPS), the ability to utilize transferrin-bound iron, and urease, have been described elsewhere (18). To date, conclusive evidence obtained by challenge experiments has been presented to confirm the role of Apx toxins and capsular material. A spontaneous Apx toxin-negative A. pleuropneumoniae strain was shown to be avirulent (14), and this result was supported later by using transposon mutagenesis (36) as well as by an isogenic A. pleuropneumoniae apxC insertion mutant (29). Also, capsule-deficient A. pleuropneumoniae strains obtained by chemical mutagenesis were shown to be attenuated (22), and this result was confirmed by reconstituting virulence properties and capsule formation upon transformation with a recombinant plasmid (39). Also, it was shown recently that the [Cu,Zn]-superoxide dismutase is not required for virulence (35). For other putative virulence factors, such as LPS (1, 3, 4), and the utilization of transferrin-bound iron (15,17,40), no conclusive challenge experiments have been performed to date. With respect to urease, data are inconclusive; ureasenegative A. pleuropneumoniae mutants hav...

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to survive on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated sequesters. It was previously demonstrated that a deletion of the anaerobic dimethyl sulfoxide reductase gene (dmsA) results in attenuation in acute disease (N. Baltes, S. Kyaw, I. Hennig-Pauka, and G. F. Gerlach, Infect. Immun. 71:6784-6792, 2003). In the present study, using two-dimensional polyacrylamide gel electrophoresis and quadrupole time-of-flight mass spectrometry, we identified an aspartate ammonia-lyase (AspA) which is upregulated upon induction with bronchoalveolar lavage fluid (BALF). This enzyme is involved in the production of fumarate, an alternative electron acceptor under anaerobic conditions. The coding gene (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 binding motif, and 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 than that of the A. pleuropneumoniae wildtype (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 the 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.

Genes expressed by Actinobacillus pleuropneumoniae in necrotic porcine lung tissue were identified by selective capture of transcribed sequences analysis. In total, 46 genes were identified, 20 of which have been previously reported to be associated with in vivo expression or virulence in A. pleuropneumoniae or in other organisms.

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 ⌬dmsA, was constructed and examined in an aerosol infection model. A. pleuropneumoniae ⌬dmsA was attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly to A. pleuropneumoniae virulence.Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is able to persist in host tissues for weeks or months after infection, surviving in tonsils as well as in sequestered necrotic lung tissue (11,14,20). In necrotic tissue, the oxygen supply is scarce, and therefore, strategies for respiration under reduced-oxygen or anaerobic conditions, such as utilization of alternative electron acceptors, are required. To date, the metabolism of A. pleuropneumoniae under these conditions has not been the subject of extensive studies, although a putative anaerobic regulator protein, HlyX, has been identified (19,31).Escherichia coli possesses an enzyme complex that allows the use of dimethyl sulfoxide (DMSO) and various other substrates as terminal electron acceptors in cell respiration under anaerobic conditions. The DMSO reductase complex, which has been extensively studied in E. coli (44), consists of three subunits, DmsA, DmsB, and DmsC. The DmsA protein (87 kDa) is the catalytic subunit containing a molybdopterin cofactor; DmsB (23 kDa) is an electron carrier containing four iron-sulfur clusters, and DmsC (30 kDa) serves as a membrane anchor for the other two subunits. The corresponding genes, dmsABC, are organized in an operon in E. coli (8). DMSO reductases have been identified in several other organisms, including Rhodobacter capsulatus (33), Rhodobacter sphaeroides (26), and Haemophilus influenzae (30). H. influenzae possesses a putative dmsABC operon containing an open reading frame (ORF) coding for a 90-kDa putative DmsA protein.In E. coli, the enzyme complex is situated at the cytoplasmic face of the inner membrane, with the DmsA and DmsB subunits facing the cytoplasm and the DmsC protein embedded in t...