Extracellular DnaK (Heat Shock Protein 70) of <i>Streptococcus mutans</i> is an endothelial modulin
DnaK (Heat Shock Protein [Hsp]70 and GroEL (Hsp60) are ubiquitous, highly conserved molecular chaperones. Bacterial HSPs have been shown to stimulate innate immunity, as well as to act as adhesins to mammalian cells. Mycobacterial Hsp70 binds to the human CD40 receptor. Streptococcus mutans is a causative agent of dental caries as well as infective endocarditis (IE). The S. mutans proteins that act as endothelial modulins and adhesins during IE remain to be completely defined. The objectives of this study were to localize extracellular S. mutans HSPs and to define functions of these proteins in interactions with endothelial cells. We identified DnaK and GroEL on the S. mutans and in culture medium through immunogold transmission electron microscopy, immunoblotting and immunofluorescence. Through sandwich enzyme-linked immunosorbent assays (ELISA) and cytokine antibody arrays, we demonstrated that recombinant S. mutans DnaK (SmDnaK), but not GroEL, can stimulate the release of proinflammatory cytokines/chemokines interleukin (IL)-6, IL-8 and growth-regulated oncogene (GRO) from human umbilical vein endothelial cells (HUVEC). This activity was enhanced upon endothelial pretreatment with interferon-gamma. Binding of SmDnaK to HUVEC was demonstrated through immunofluorescence microscopy, and increased binding was seen upon interferon-gamma pretreatment. Antibody blocking of DnaK on the surface of S. mutans decreased bacterial adherence to HUVEC, as evidenced by microscopy and flow cytometry. Expression of CD40 in HEK293 cells did not support increased adherence of SmDnaK. Additionally, SmDnaK failed to stimulate cytokine expression from CD40-expressing HEK293 cells. We conclude that S. mutans expresses DnaK and GroEL on its surface, as well as releases these HSPs into the culture medium. S. mutans DnaK, but not GroEL, can act as an endothelial modulin through recognition of an interferon-gamma-inducible receptor. The cellular binding of S. mutans DnaK is most likely CD40-independent. DnaK contributes to the adherence of S. mutans to endothelial cells. Our work suggests that DnaK is a modulin of the host immune response to S. mutans, and it might be utilized as a virulence factor during S. mutans attachment in the initial stages of IE.