A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Identification of a Novel Bacterial Outer Membrane Interleukin-1B-Binding Protein from Aggregatibacter actinomycetemcomitans
Tekijät: Paino A, Ahlstrand T, Nuutila J, Navickaite I, Lahti M, Tuominen H, Välimaa H, Lamminmäki U, Pöllänen MT, Ihalin R
Kustantaja: PUBLIC LIBRARY SCIENCE
Julkaisuvuosi: 2013
Journal: PLoS ONE
Tietokannassa oleva lehden nimi: PLOS ONE
Lehden akronyymi: PLOS ONE
Artikkelin numero: UNSP e70509
Numero sarjassa: 7
Vuosikerta: 8
Numero: 7
Sivujen määrä: 15
ISSN: 1932-6203
DOI: https://doi.org/10.1371/journal.pone.0070509
Tiivistelmä
Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1 beta. Some bacterial species can alter their physiological properties as a result of sensing IL-1 beta. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1 beta sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1 beta through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1 beta than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1 beta, but this binding was not specific, as a control protein for IL-1 beta also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1 beta-binding surface-exposed lipoprotein that may be part of the bacterial IL-1 beta-sensing system.
Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1 beta. Some bacterial species can alter their physiological properties as a result of sensing IL-1 beta. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1 beta sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1 beta through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1 beta than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1 beta, but this binding was not specific, as a control protein for IL-1 beta also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1 beta-binding surface-exposed lipoprotein that may be part of the bacterial IL-1 beta-sensing system.
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