IADR Abstract Archives
Interaction of Aggregatibacter actinomycetemcomitans with Dendritic cells
Objectives: Pathogens have evolved survival strategies to avoid or withstand the host immune response. One mechanism is to adapt to a temporary or permanent intracellular life style, which impedes the surveillance of the immune system. Dendritic cells (DCs), which play a sensor and sentinel role for invading pathogens, are important for maintaining the gingival immune homeostasis. The extracellular matrix protein adhesin A (EmaA) of Aggregatibacter actinomycetemcomitans is a glycoprotein that trimerizes to form antenna-like structures on the surface of the bacterium. We propose that EmaA structures interact with C-type lectin receptors of DCs. The objective of this study is to determine the EmaA-mediated interaction of DCs with A. actinomycetemcomitans.
Methods: Mouse bone marrow-derived DCs were isolated and co-cultured with wild-type (WT) and emaA- strains expressing Green Florescent Protein (GFP). Co-cultures were analyzed by live imaging or high-resolution confocal laser scanning microscopy (CLSM). Bacterial entry kinetics were determined using Flow cytometry and a gentamycin protection assay to quantify live intracellular bacteria.
Results: Live imaging of DCs co-cultured with GFP-A. actinomycetemcomitans revealed bacterial internalization within thirty minutes and bacterial replication inside of the DCs. CLSM supported A. actinomycetemcomitans internalization and the cytoplasmic location of infected DCs. A steady increase of bacterial fluorescence intensity over time was observed, which can be inhibited by the addition of Cytochalasin D, a phagocytosis inhibitor. A dramatic decrease in the fluorescent intensity of emaA- compared with the WT strain was observed by flow cytometry and this reduction was verified using the quantitative gentamycin protection assay.
Conclusions: Our studies suggest that A. actinomycetemcomitans has co-evolved mechanisms for the interaction and persistence within DCs. The internalization of A. actinomycetemcomitans is mediated by EmaA structures. Our novel finding of the internalization and persistence of A. actinomycetemcomitans in DCs is an important mechanism utilized by this pathogen to survive and possibly disseminate to non-oral tissues.
Methods: Mouse bone marrow-derived DCs were isolated and co-cultured with wild-type (WT) and emaA- strains expressing Green Florescent Protein (GFP). Co-cultures were analyzed by live imaging or high-resolution confocal laser scanning microscopy (CLSM). Bacterial entry kinetics were determined using Flow cytometry and a gentamycin protection assay to quantify live intracellular bacteria.
Results: Live imaging of DCs co-cultured with GFP-A. actinomycetemcomitans revealed bacterial internalization within thirty minutes and bacterial replication inside of the DCs. CLSM supported A. actinomycetemcomitans internalization and the cytoplasmic location of infected DCs. A steady increase of bacterial fluorescence intensity over time was observed, which can be inhibited by the addition of Cytochalasin D, a phagocytosis inhibitor. A dramatic decrease in the fluorescent intensity of emaA- compared with the WT strain was observed by flow cytometry and this reduction was verified using the quantitative gentamycin protection assay.
Conclusions: Our studies suggest that A. actinomycetemcomitans has co-evolved mechanisms for the interaction and persistence within DCs. The internalization of A. actinomycetemcomitans is mediated by EmaA structures. Our novel finding of the internalization and persistence of A. actinomycetemcomitans in DCs is an important mechanism utilized by this pathogen to survive and possibly disseminate to non-oral tissues.