Cloning and characterisation of type 4 fimbrial genes from Actinobacillus pleuropneumoniae
Andrew Stevenson1, Julie Macdonald, Mark Roberts
1Molecular Bacteriology Group, Department of Veterinary Pathology, Glasgow University Veterinary School, Bearsden Road, Glasgow G61 1QH, Scotland, UK.
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.
Insights
This study identifies the type 4 fimbrial gene (apfA) in Actinobacillus pleuropneumoniae, revealing conserved features important for bacterial adherence and host colonization in pigs.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, but colonization mechanisms are poorly understood.
- Fimbriae are bacterial appendages involved in adherence to host tissues.
- Fimbriae have been observed on Actinobacillus pleuropneumoniae, suggesting a role in pathogenesis.
Purpose of the Study:
- To identify and characterize the type 4 fimbrial structural gene (apfA) in Actinobacillus pleuropneumoniae.
- To investigate the homology of other identified open reading frames with known type 4 fimbrial biogenesis genes.
- To analyze the conserved features of the ApfA protein and its potential role in bacterial adhesion.
Main Methods:
- Identification and sequencing of the apfA gene from Actinobacillus pleuropneumoniae.
- Bioinformatic analysis to identify homologous genes and predict protein structure.
- Comparison of ApfA sequence with other type 4 fimbriae, particularly from HAP organisms.
- Analysis of apfA gene conservation across different serotypes of Actinobacillus pleuropneumoniae.
- Production of recombinant ApfA and generation of specific antisera.
Main Results:
- The type 4 fimbrial structural gene (apfA) was identified and characterized in Actinobacillus pleuropneumoniae.
- Homologous genes involved in type 4 fimbrial biogenesis, including a putative leader specific peptidase (apfD), were found.
- The predicted ApfA polypeptide shares conserved residues with other group A type 4 fimbriae, including a unique alanine at the cleavage site.
- A conserved motif in the predicted loop of ApfA is shared with type 4 fimbriae of other HAP organisms.
- The apfA gene is present and conserved across various serotypes of Actinobacillus pleuropneumoniae.
- Recombinant ApfA was successfully produced and used to generate specific antibodies.
Conclusions:
- The identification of apfA and related genes provides insights into the type 4 fimbrial system of Actinobacillus pleuropneumoniae.
- Conserved features of ApfA suggest a significant role in bacterial adherence and colonization of the porcine respiratory tract.
- The findings contribute to understanding the pathogenesis of porcine pleuropneumonia.
- Further research into the function of these fimbriae could lead to novel strategies for disease control.
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