Streptococcal Protein Fragment Complementation (S-PFC) to Detect Protein-Protein Interactions

Streptococcus mutans is a major pathogen responsible for dental caries but over one third of its gene products are annotated as hypothetical proteins. To transform the genomic data into functional information we need to map the unknown hypothetical proteins to known biological pathways by protein-protein interaction analysis. OBJECTIVE: To develop a simple and rapid method to assay protein-protein interactions in S. mutans. We have termed this method Streptococcal Protein Fragment Complementation (S-PFC). METHODS: S-PFC is based on bacterial resistance to antibiotic trimethoprim conferred by murine dihydrofolate reductase (mDHFR). We have manipulated mDHFR to split the enzyme into two separate domains; F[12] & F[3]. When F[12] & F[3] are closely associated with each other, they reconstitute a fully functional mDFHR. These new domains can be linked to putative interacting proteins, where a definitive protein-protein interaction would reconstitute a fully functional mDFHR, thus providing antibiotic resistance to trimethoprim. RESULTS: We demonstrated the viability of this system in S. mutans by utilizing the homodimerizing ability of a yeast transcriptional activator GCN4. Fragments F[12] and F[3] were independently linked to GCN4 and placed at separate locations on a plasmid. In trans, the fragment linked GCN4 gene products reconstituted a fully functional mDFHR and resistance to trimethoprim. CONCLUSION: We have developed S-PFC system that allows for the detection of protein-protein interactions in streptococcal cells. This method will help to map functionally unknown proteins to known biological processes. The potential for S-PFC to be an invaluable molecular biology tool is far reaching and bring us one step closer to fully elucidating protein-protein interactions in S. mtuans.

This work was supported by NIH DE014726 grant.