Enzymes Involved in Anaerobic Respiration Appear To Play a Role inActinobacillus pleuropneumoniaeVirulence

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Abstract
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 allA. pleuropneumoniaeserotype reference strains. The transcriptional start point was identified downstream of a putative FNR binding motif, and BALF-dependent activation ofaspAwas confirmed by construction of an isogenicA. pleuropneumoniaemutant carrying a chromosomalaspA::luxABtranscriptional fusion. TwoaspAdeletion mutants,A. pleuropneumoniaeΔaspAandA. pleuropneumoniaeΔaspAΔdmsA, 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 theA. pleuropneumoniaewild-type (wt) controls. Pigs challenged withA. pleuropneumoniaeΔaspAΔdmsAhad a significantly lower clinical score, and this mutant was rarely reisolated from unaltered lung tissue; in contrast,A. pleuropneumoniaeΔaspAand theA. pleuropneumoniaewt 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 inA. pleuropneumoniaepathogenesis.

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