A targeted mutagenesis system for Actinobacillus pleuropneumoniae

Area of Science:

• Microbiology
• Bacteriology
• Molecular Biology

Background:

• Actinobacillus pleuropneumoniae (Ap) causes contagious pleuropneumonia in swine.
• Understanding the roles of specific Ap genes, such as those encoding outer membrane proteins, is crucial for disease control.

Purpose of the Study:

• To develop and validate methods for creating isogenic mutants of Ap.
• To construct Ap mutants deficient in the 48-kDa outer membrane protein (AopA) for further pathogenesis studies.

Main Methods:

• Gene mutagenesis of cloned Ap genes.
• Allelic exchange for constructing Ap mutants.
• Insertion of a kanamycin resistance gene (KmR) to inactivate the aopA locus.
• Use of a conjugative, lambda pir-dependent suicide vector for gene transfer.
• Filter mating technique for introducing the vector into Ap.
• Southern and Western blot analyses to confirm allelic replacement and protein absence.

Main Results:

• Successfully constructed isogenic Ap mutants lacking the AopA protein.
• Demonstrated allelic replacement of the native aopA locus with a mutated, kanamycin-resistant locus.
• Confirmed the absence of AopA production in constructed mutants.
• Achieved successful mutant construction in Ap serotypes 1 and 5.

Conclusions:

• The described methods are effective for generating Ap mutants with inactivated genes.
• These techniques provide a valuable tool for analyzing the function of Ap genes in swine pleuropneumonia.
• The AopA- mutants are suitable for investigating the protein's role in Ap pathogenesis.