Surface display of Aggregatibacter actinomycetemcomitans autotransporter Aae and dispersin B hybrid act as antibiofilm agents.
Among the various proteins expressed by the periodontopathogen Aggregatibacter actinomycetemcomitans, two proteins play important roles for survival in the oral cavity. The autotransporter Aae facilitates the attachment of the pathogen to oral epithelial cells, which act as a reservoir, while the biofilm-degrading glycoside hydrolase dispersin B facilitates the movement of daughter cells from the mature biofilm to a new site. The objective of this study was to use the potential of these two proteins to control biofilms. To this end, we generated a hybrid construct between the Aae C-terminal translocating domain and dispersin B, and mobilized it into Escherichia coli Rosetta (DE3) pLysS cells. Immunofluorescence analysis of the modified E. coli cells confirmed the presence of dispersin B on the surface. Further, the membrane localization of the displayed dispersin B was confirmed with Western blot analysis. The integrity of the E. coli cells displaying the dispersin B was confirmed through FACS analysis. The hydrolytic activity of the surface-displayed dispersin B was confirmed by using 4-methylumbelliferyl-β-d-glucopyranoside as the substrate. The detachment ability of the dispersin B surface-displaying E. coli cells was shown using Staphylococcus epidermidis and Actinobacillus pleuropneumoniae biofilms in a microtiter assay. We concluded that the Aae β-domain is sufficient to translocate foreign enzymes in the native folded form and that the method of Aae-mediated translocation of surface displayed enzymes might be useful for control of biofilms.
Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
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Ragunath
Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
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DiFranco
Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
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Shanmugam
Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
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Gopal
Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
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Vyas
Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
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Fine
Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
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Cugini
Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
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Ramasubbu
D016977:Actinobacillus pleuropneumoniae / Q000502:physiology; D016976:Aggregatibacter actinomycetemcomitans / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development*; D061505:Cell Surface Display Techniques; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013212:Staphylococcus epidermidis / Q000502:physiology; D000069320:Type V Secretion Systems / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000069320:Type V Secretion Systems; D006026:Glycoside Hydrolases; C501330:dispersin B, Actinobacillus actinomycetemcomitans
autotransporter; biofilm; dispersin B; oral pathogen; surface display