Identification of Actinobacillus pleuropneumoniae virulence genes using signature-tagged mutagenesis in a swine infection model

Fuller, Troy E.; Martin, Stephen; Teel, Janet F.; Alaniz, G.; Kennedy, Michael J.; Lowery, David E.

doi:10.1006/mpat.2000.0364

Cited by 66 publications

(64 citation statements)

References 42 publications

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“…In total we found 105 of 2,064 (5.1%) mutants to be attenuated, with 55 different genes disrupted. In a previous STM screen of A. pleuropneumoniae (29), 110 of 800 (13.8%) potentially attenuated mutants were found, but only 20 different genes were identified, and of these only 3 (tig, pnp, and apvD/macA) are common to that study and ours. In addition, only one of the genes (mrp) identified in an in vivo expression technology study of A. pleuropneumoniae (30) was also found in our STM screen.…”

Section: Pleuropneumoniae Genes Important In Vivo 3965supporting

confidence: 49%

“…This inconsistency was overcome by increasing the challenge dose to 10 6 to 10 7 CFU/ pig. In an earlier STM study of A. pleuropneumoniae infection in pigs (29), bacteria were recovered by lung lavage following experimental infection. However, we found this method to be unreliable, yielding essentially random recovery of individual signature tags.…”

Section: Resultsmentioning

confidence: 99%

“…In P. multocida, all eight subunits of the bacterial enzyme are encoded by the atp operon, with atpA flanked by atpH and atpG (61). This gene order is the same in A. pleuropneumoniae, and both atpH and atpG have been previously implicated in the pathogenesis of A. pleuropneumoniae infection (29).…”

Section: Resultsmentioning

confidence: 99%

“…In addition to their fundamental roles in protein folding and translocation, DnaK and DnaJ constitute the primary stress-sensing and transduction systems of the E. coli heat shock response (95) where they modulate the induction of the heat shock response by altering the stability of the heat shock sigma factor 32 . Significantly, mutations in DnaK have previously been shown to attenuate A. pleuropneumoniae virulence, and to decrease the survival of Brucella suis in human macrophage-like cell lines (29,52). Strain 26B6 contains an insertion in an htpG gene homologue.…”

Section: Pleuropneumoniae Genes Important In Vivo 3965mentioning

confidence: 99%

“…It has been suggested that HtpG serves as a holder chaperone, transiently maintaining a subset of de novo synthesized proteins in a conformation that is accessible to DnaK and DnaJ (26). In strain 22A10, the transposon has inserted in a trigger factor (tig) gene homologue that was identified in a previous STM study of A. pleuropneumoniae (29). Trigger factor is a molecular chaperone with peptidyl-prolyl isomerase activity.…”

Section: Pleuropneumoniae Genes Important In Vivo 3965mentioning

confidence: 99%

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

et al. 2003

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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.

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Section: Pleuropneumoniae Genes Important In Vivo 3965supporting

confidence: 49%

Section: Resultsmentioning

confidence: 99%