Identification ofActinobacillus pleuropneumoniaeGenes Important for Survival during Infection in Its Natural Host

Abstract

Actinobacillus pleuropneumoniae is a strict respiratory tract pathogen of swine and is the causative agent of porcine pleuropneumonia. We have used signature-tagged mutagenesis (STM) to identify genes required for survival of the organism within the pig. A total of 2,064 signature-tagged Tn10 transposon mutants were assembled into pools of 48 each, and used to inoculate pigs by the endotracheal route. Out of 105 mutants that were consistently attenuated in vivo, only 11 mutants showed a >2-fold reduction in growth in vitro compared to the wild type, whereas 8 of 14 mutants tested showed significant levels of attenuation in pig as evidenced from competitive index experiments. Inverse PCR was used to generate DNA sequence of the chromosomal domains flanking each transposon insertion. Only one sibling pair of mutants was identified, but three apparent transposon insertion hot spots were found-an anticipated consequence of the use of a Tn10-based system. Transposon insertions were found within 55 different loci, and similarity (BLAST) searching identified possible analogues or homologues for all but four of these. Matches included proteins putatively involved in metabolism and transport of various nutrients or unknown substances, in stress responses, in gene regulation, and in the production of cell surface components. Ten of the sequences have homology with genes involved in lipopolysaccharide and capsule production. The results highlight the importance of genes involved in energy metabolism, nutrient uptake and stress responses for the survival of A. pleuropneumoniae in its natural host: the pig.

Regulation of pga Operon Expression and Biofilm Formation in Actinobacillus pleuropneumoniae by σ E and H-NS

“…A. pleuropneumoniae S4074 T (here designated wild type [WT]) and its derivatives 5B3 and 19B10 (49), which contain Tn10 transposon insertions in hns and rseA (mclA), respectively, were used in this study. An additional 60 adherent mutants from the Sheehan et al (49) STM screen and a serotype 1 clinical isolate (M2000) were also investigated. Bacteria were grown at 37°C in 5% CO 2 in brain heart infusion (BHI) agar (Difco) supplemented with 1 g/ml ␤-NAD (BHI-NAD agar) or in BHI-NAD broth.…”

“…Sequences of all primers used for PCRs are listed in Table 1, and those for quantitative reverse transcription-PCR (qRT-PCR) are listed in Table 2. Locations of Tn10 insertions were determined by inverse PCR and sequencing as previously described (49), except that chromosomal DNA was obtained using the FastDNA Spin kit, according to the manufacturer's instructions (Qbiogene, United Kingdom). Mutations in hns were confirmed by direct PCR using one of the two hns-specific primers (hns_F or hns_R) in combination with the Tn10-specific primer BS401 (49).…”

“…In order to determine if the phenotype associated with the rseA mutation was due to deregulation of RpoE, the rpoE gene was amplified by PCR (using primers rpoEExp_F and rpoEExp_R) and cloned into the vector pMC-Express (8) under the control of the sodC promoter of A. pleuropneumoniae, which confers constitutive expression. The resulting plasmid, pMCrpoE, was conjugated into S4074 T , as previously described (49).…”

“…Interestingly, only 1 of these mutants (previously identified as mclA::Km) was attenuated for acute infection in pigs (49), whereas the other 61 mutants were recovered from infected lungs, indicating no loss of virulence during acute infection. The mclA (also called rseA [regulator of sigma E]) gene encodes an anti-sigma factor that negatively regulates activity of the extracytoplasmic stress response sigma factor RpoE/ E (1).…”

“…This bacterium is a strict parasite of the porcine respiratory tract and is most often recovered from sequestered lung lesions and/or from tonsils of chronically infected pigs and is recovered less frequently from the nasal cavities (14,15,60). Many of the virulence factors that contribute to the ability of A. pleuropneumoniae to cause acute disease have been identified (23,49). However, less is known about the factors that contribute to this bacterium's ability to persist and cause chronic infection.…”