Streptococcus mutans autolysin AtlA is a fibronectin-binding protein and contributes to bacterial survival in the bloodstream and virulence for infective endocarditis.

Streptococcus mutans, a commensal of the human oral cavity, can survive in the bloodstream and cause infective endocarditis (IE). However, the virulence factors associated with this manifestation of disease are not known. Here, we demonstrate that AtlA, an autolysin of S. mutans is a newly identified fibronectin (Fn) binding protein and contributes to bacterial resistance to phagocytosis and survival in the bloodstream. Interestingly, prior exposure to plasma at low concentrations was sufficient to enhance bacterial survival in the circulation. Calcium ions at physiological plasma concentrations induced maturation of AtlA from the 104-90 kDa isoform resulting in increased Fn binding and resistance to phagocytosis. An isogenic mutant strain defective in AtlA expression exhibited reduced survival and virulence when tested in a rat model of IE compared with the wild-type and complemented strains. The data presented suggest that plasma components utilized by S. mutans enhanced survival in the circulation and AtlA is a virulence factor associated with infective endocarditis.

Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication.

Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication.