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Infection and Immunity, July 2003, p. 3960-3970, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.3960-3970.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of Actinobacillus pleuropneumoniae Genes Important for Survival during Infection in Its Natural Host

Brian J. Sheehan,1,{dagger} Janine T. Bossé,1 Amanda J. Beddek,2 Andrew N. Rycroft,2 J. Simon Kroll,1 and Paul R. Langford1*

Department of Paediatrics, Imperial College London, St. Mary's Campus, London W2 1PG,1 Department of Pathology and Infectious Diseases, The Royal Veterinary College, North Mymms, Hatfield, Herts AL9 7TA, United Kingdom2

Received 10 February 2003/ Returned for modification 13 March 2003/ Accepted 2 April 2003

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.

* Corresponding author. Mailing address: Department of Paediatrics, Imperial College London, St. Mary's Campus, London W2 1PG, United Kingdom. Phone: 44-207-886-6340. Fax: 44-207-886-6284. E-mail: p.langford{at}imperial.ac.uk.

Editor: J. N. Weiser

{dagger} Present address: Intervet, Walton Manor, Milton Keynes, Bucks MK7 7AJ, United Kingdom.

Infection and Immunity, July 2003, p. 3960-3970, Vol. 71, No. 7
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.7.3960-3970.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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