Limits...
A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1 β and interleukin-8

View Article: PubMed Central - PubMed

ABSTRACT

Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

No MeSH data available.


Related in: MedlinePlus

BilRI bound to various human inflammatory cytokines but not to fibrinogen or collagen. A) Recombinant BilRI containing an 8-histidine-long C-terminal tag bound to various recombinant human cytokines in a microplate assay. BSA served as a negative control and was used as a blocking agent in the assays. The bound BilRI was detected with HRP-labeled HisProbe™. The BilRI binding to IL-8 was high compared to the binding to the control protein BSA (**:p = 0.008, paired-samples T-test). B) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to fibrinogen-coated wells in a microplate assay when detected with europium-labeled antibody against the histidine tag. Although the positive control recombinant ClfA of S. aureus bound fibrinogen efficiently, the recombinant FgbA, which has been reported to bind fibrinogen,42 did not show positive binding in the assay. Only BilRI binding to IL-8 showed a statistically significant positive difference compared with the BSA control (p = 0.028, Mann-Whitney U-test) from the test experiments. C) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to collagen-coated wells in the microplate assays when detected with europium-labeled antibody against the histidine tag. The positive control recombinant YadA of Y. enterocolitica bound collagen efficiently. Only BilRI binding IL-8 showed a statistically significant positive difference compared with control BSA (p = 0.028, Mann-Whitney U-test) from the test experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5383217&req=5

f0002: BilRI bound to various human inflammatory cytokines but not to fibrinogen or collagen. A) Recombinant BilRI containing an 8-histidine-long C-terminal tag bound to various recombinant human cytokines in a microplate assay. BSA served as a negative control and was used as a blocking agent in the assays. The bound BilRI was detected with HRP-labeled HisProbe™. The BilRI binding to IL-8 was high compared to the binding to the control protein BSA (**:p = 0.008, paired-samples T-test). B) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to fibrinogen-coated wells in a microplate assay when detected with europium-labeled antibody against the histidine tag. Although the positive control recombinant ClfA of S. aureus bound fibrinogen efficiently, the recombinant FgbA, which has been reported to bind fibrinogen,42 did not show positive binding in the assay. Only BilRI binding to IL-8 showed a statistically significant positive difference compared with the BSA control (p = 0.028, Mann-Whitney U-test) from the test experiments. C) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to collagen-coated wells in the microplate assays when detected with europium-labeled antibody against the histidine tag. The positive control recombinant YadA of Y. enterocolitica bound collagen efficiently. Only BilRI binding IL-8 showed a statistically significant positive difference compared with control BSA (p = 0.028, Mann-Whitney U-test) from the test experiments.

Mentions: A microplate assay showed that recombinant BilRI bound to various cytokines, of which the binding to IL-8 was high compared with the binding of BilRI to the negative control protein bovine serum albumin (BSA; p = 0.008; paired-samples T-test; Fig. 2A). However, the binding to IL-6-coated wells was weak and almost as inefficient as the binding to BSA, which was used as a blocking agent in the assay (Fig. 2A). We decided to use C-tagged recombinant BilRI in our binding assays because binding to IL-1β was originally shown with a similar protein.24 However, we also tested an N-tagged variant of BilRI, which did not show increased binding to IL-1β, IL-8, or IL-6 compared with the C-tagged protein (data not shown). BilRI did not bind to fibrinogen- (Fig. 2B) or to collagen (Fig. 2C)-coated wells. Moreover, BilRI binding to IL-8 was weaker than the fibrinogen-binding of positive control protein clumping factor A (ClfA) of Staphylococcus aureus and the collagen-binding of positive control protein YadA of Yersinia enterocolitica (Fig. 2C).Figure 2.


A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1 β and interleukin-8
BilRI bound to various human inflammatory cytokines but not to fibrinogen or collagen. A) Recombinant BilRI containing an 8-histidine-long C-terminal tag bound to various recombinant human cytokines in a microplate assay. BSA served as a negative control and was used as a blocking agent in the assays. The bound BilRI was detected with HRP-labeled HisProbe™. The BilRI binding to IL-8 was high compared to the binding to the control protein BSA (**:p = 0.008, paired-samples T-test). B) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to fibrinogen-coated wells in a microplate assay when detected with europium-labeled antibody against the histidine tag. Although the positive control recombinant ClfA of S. aureus bound fibrinogen efficiently, the recombinant FgbA, which has been reported to bind fibrinogen,42 did not show positive binding in the assay. Only BilRI binding to IL-8 showed a statistically significant positive difference compared with the BSA control (p = 0.028, Mann-Whitney U-test) from the test experiments. C) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to collagen-coated wells in the microplate assays when detected with europium-labeled antibody against the histidine tag. The positive control recombinant YadA of Y. enterocolitica bound collagen efficiently. Only BilRI binding IL-8 showed a statistically significant positive difference compared with control BSA (p = 0.028, Mann-Whitney U-test) from the test experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383217&req=5

f0002: BilRI bound to various human inflammatory cytokines but not to fibrinogen or collagen. A) Recombinant BilRI containing an 8-histidine-long C-terminal tag bound to various recombinant human cytokines in a microplate assay. BSA served as a negative control and was used as a blocking agent in the assays. The bound BilRI was detected with HRP-labeled HisProbe™. The BilRI binding to IL-8 was high compared to the binding to the control protein BSA (**:p = 0.008, paired-samples T-test). B) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to fibrinogen-coated wells in a microplate assay when detected with europium-labeled antibody against the histidine tag. Although the positive control recombinant ClfA of S. aureus bound fibrinogen efficiently, the recombinant FgbA, which has been reported to bind fibrinogen,42 did not show positive binding in the assay. Only BilRI binding to IL-8 showed a statistically significant positive difference compared with the BSA control (p = 0.028, Mann-Whitney U-test) from the test experiments. C) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to collagen-coated wells in the microplate assays when detected with europium-labeled antibody against the histidine tag. The positive control recombinant YadA of Y. enterocolitica bound collagen efficiently. Only BilRI binding IL-8 showed a statistically significant positive difference compared with control BSA (p = 0.028, Mann-Whitney U-test) from the test experiments.
Mentions: A microplate assay showed that recombinant BilRI bound to various cytokines, of which the binding to IL-8 was high compared with the binding of BilRI to the negative control protein bovine serum albumin (BSA; p = 0.008; paired-samples T-test; Fig. 2A). However, the binding to IL-6-coated wells was weak and almost as inefficient as the binding to BSA, which was used as a blocking agent in the assay (Fig. 2A). We decided to use C-tagged recombinant BilRI in our binding assays because binding to IL-1β was originally shown with a similar protein.24 However, we also tested an N-tagged variant of BilRI, which did not show increased binding to IL-1β, IL-8, or IL-6 compared with the C-tagged protein (data not shown). BilRI did not bind to fibrinogen- (Fig. 2B) or to collagen (Fig. 2C)-coated wells. Moreover, BilRI binding to IL-8 was weaker than the fibrinogen-binding of positive control protein clumping factor A (ClfA) of Staphylococcus aureus and the collagen-binding of positive control protein YadA of Yersinia enterocolitica (Fig. 2C).Figure 2.

View Article: PubMed Central - PubMed

ABSTRACT

Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

No MeSH data available.


Related in: MedlinePlus