title: Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae.
abstract: Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumoniae. Little is known about the mechanisms by which A. pleuropneumoniae colonises the respiratory tract. Fimbriae are common mediators of bacterial adherence to mucosal epithelia and have been observed on the surface of A. pleuropneumoniae cells. Here we report the identification and characterisation of the type 4 fimbrial structural gene (apfA) from A. pleuropneumoniae. In addition a number of open reading frames were identified in A. pleuropneumoniae that have significant homology to type 4 fimbrial biogenesis genes from other species, including a putative leader specific peptidase (apfD). A. pleuropneumoniae apfA codes for a predicted polypeptide of approximately 16kDa, removal of the leader sequence at the predicted cleavage site would yield a 14.5kDa polypeptide. The first 30 residues of the mature polypeptide are well conserved with other members of the group A type 4 fimbriae family. The signal sequence of ApfA is 13 amino acids in length and, unusually, the residue that precedes the cleavage site is alanine rather than glycine which is found in most other type 4 fimbriae. The C-terminus of ApfA possesses cysteine residues that are conserved in type 4 fimbriae of many species. In other type 4 fimbriae the distal C-terminal cysteines form a disulphide bond that produces a loop, which is important for the function of fimbriae and also comprises a major antigenic determinant. A motif within the predicted loop in ApfA was found to be highly conserved in type 4 fimbriae of other HAP organisms (Haemophilus, Actinobacillus, Pasteurella). The A. pleuropneumoniae type 4 fimbrial biogenesis genes showed the strongest homology to putative type 4 fimbrial genes of Haemophilus ducreyi. A. pleuropneumoniae apfA gene was shown to be present and highly conserved in different serotypes of A. pleuropneumoniae. Recombinant ApfA was produced and used to raise anti-ApfA antisera.
authors.affiliation: Molecular Bacteriology Group, Department of Veterinary Pathology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, Scotland, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Stevenson
authors.forename: Julie
authors.initials: J
authors.lastname: Macdonald
authors.forename: Mark
authors.initials: M
authors.lastname: Roberts
mesh_terms: D016977:Actinobacillus pleuropneumoniae / Q000235:genetics* / Q000502:physiology; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D010861:Fimbriae, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D009876:Operon / Q000235:genetics; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D000907:Antibodies, Bacterial; D004269:DNA, Bacterial; D011994:Recombinant Proteins