IAI Accepts, published online ahead of print on 13 October 2008

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Infect. Immun. doi:10.1128/IAI.00085-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Functional characterization of AasP, a maturation protease autotransporter protein of Actinobacillus pleuropneumoniae

Tehmeena Ali, Neil J. Oldfield, Karl G. Wooldridge, David P. Turner, and Dlawer A. Ala'Aldeen^*

Molecular Bacteriology and Immunology Group, Institute of Infection, Immunity & Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, United Kingdom

^* To whom correspondence should be addressed. Email: daa{at}nottingham.ac.uk.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia: a highly contagious respiratory infection of pigs. AasP, a putative subtilisin-like serine protease autotransporter, has recently been identified in A. pleuropneumoniae. We hypothesized that similar to other autotransporters of this type AasP may undergo autocatalytic cleavage resulting in release of the passenger domain of the protein. Furthermore, AasP may be responsible for the cleavage of other A. pleuropneumoniae outer membrane proteins. To address these hypotheses, the aasP gene was cloned and the expressed recombinant AasP protein used to raise mono-specific rabbit anti-serum. Immunoblot analysis of whole cell lysates and secreted proteins demonstrated that AasP does not undergo proteolytic cleavage. Immunoblot analysis also confirmed that AasP is universally expressed by A. pleuropneumoniae. Confirmation of the maturation protease function of AasP was obtained through phenotypic analysis of an A. pleuropneumoniae aasP deletion mutant and by functional complementation. Comparison of the secreted proteins of the wild type, an aasP mutant derivative and an aasP mutant complemented in trans, led to the identification of OmlA protein fragments that were only present in the secreted protein preparations from the wild type and complemented strains indicating that AasP is involved in the modification of OmlA. This is the first demonstration of a function for any autotransporter protein in Actinobacillus pleuropneumoniae.