Binding of porcine plasma ficolin-alpha and mannose-binding lectin A to biofilm cultures of Actinobacillus pleuropneumoniae

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Binding of porcine plasma ficolin-alpha and mannose-binding lectin A to biofilm cultures of Actinobacillus pleuropneumoniae

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dc.contributor.advisor Turner, Patricia
dc.contributor.advisor Hayes, Tony
dc.contributor.author Puttaswamy, Anil
dc.date 2012-04-05
dc.date.accessioned 2012-04-19T20:30:32Z
dc.date.available 2012-04-19T20:30:32Z
dc.date.issued 2012-04-19
dc.identifier.uri http://hdl.handle.net/10214/3508
dc.description.abstract Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. en_US
dc.description.sponsorship Natural Sciences and Engineering Research Council of Canada (NSERC) en_US
dc.language.iso en en_US
dc.subject Mannose-binding lectin, Mannan-binding lectin, ficolin, Actinobacillus pleuropneumoniae, N-acetylglucosamine, poly N-acetylglucosamine, biofilm en_US
dc.title Binding of porcine plasma ficolin-alpha and mannose-binding lectin A to biofilm cultures of Actinobacillus pleuropneumoniae en_US
dc.type Thesis en_US
dc.degree.programme Pathobiology en_US
dc.degree.name Doctor of Veterinary Science en_US
dc.degree.department Department of Pathobiology en_US


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