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Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria
Published on: September 16, 2013
Isolation and characterization of LPS mutants of Actinobacillus pleuropneumoniae serotype 1
S Rioux1, C Bégin, J D Dubreuil
1Groupe de Recherche sur les Maladies Infectieuses du Porc, and Département de pathologie et microbiologie, Faculté de médecine vétérinaire, Université de Montréal, C.P. 5000, St-Hyacinthe (Québec), Canada J2S 7C6.
Abstract:
The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae.
Insights
Lipopolysaccharide (LPS) from Actinobacillus pleuropneumoniae is a key virulence factor. Mutants lacking specific LPS structures showed reduced adherence and significantly lower virulence in animal models.
Area of Science:
- Microbiology
- Immunology
- Animal Health
Background:
- Actinobacillus pleuropneumoniae causes swine pleuropneumonia.
- Lipopolysaccharide (LPS) is identified as a major adhesin and virulence factor.
Purpose of the Study:
- To isolate and characterize LPS mutants of A. pleuropneumoniae.
- To investigate the role of LPS in A. pleuropneumoniae virulence.
Main Methods:
- Colony dot screening and novobiocin sensitivity assays were used to identify LPS mutants.
- SDS-PAGE and Western blot analysis characterized LPS structure.
- Adherence to piglet trachea and virulence in mice were assessed.
Main Results:
- Two mutants (F19 and 33.1) lacked high-molecular-mass LPS bands and O-antigen reactivity.
- Mutant F19 showed similar tracheal adherence, while mutant 33.1 showed reduced adherence compared to the parent strain.
- Both LPS mutants exhibited significantly reduced virulence in a mouse model.
Conclusions:
- LPS plays a crucial role in the virulence of A. pleuropneumoniae.
- Specific LPS structures are important for bacterial adherence and pathogenicity.
