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Infection and Immunity, January 2005, p. 226-234, Vol. 73, No. 1
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.1.226-234.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Enzymes Involved in Anaerobic Respiration Appear To Play a Role in Actinobacillus pleuropneumoniae Virulence

Department of Infectious Diseases, Institute for Microbiology,¹ Clinic for Pigs and Small Ruminants, University of Veterinary Medicine, Foundation, Hannover,² Department of Cell Biology, German Research Centre for Biotechnology (GBF), Braunschweig, Germany³

Received 23 June 2004/ Returned for modification 4 August 2004/ Accepted 3 September 2004

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 aspAdmsA, 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 wild-type (wt) controls. Pigs challenged with A. pleuropneumoniae aspAdmsA 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.

Editor: F. C. Fang

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