Functional characterization of AasP, a maturation protease autotransporter protein of Actinobacillus pleuropneumoniae.
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a highly contagious respiratory infection in pigs. AasP, a putative subtilisin-like serine protease autotransporter, has recently been identified in A. pleuropneumoniae. We hypothesized that, similarly 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 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 monospecific rabbit antiserum. 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 present only in the secreted-protein preparations of the wild-type and complemented strains, indicating that AasP is involved in modification of OmlA. This is the first demonstration of a function for any autotransporter protein in Actinobacillus pleuropneumoniae.
Molecular Bacteriology and Immunology Group, Institute of Infection, Immunity & Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom.
Tehmeena
T
Ali
Neil J
NJ
Oldfield
Karl G
KG
Wooldridge
David P
DP
Turner
Dlawer A A
DA
Ala'Aldeen
D016977:Actinobacillus pleuropneumoniae / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases