Disulfide bond formation in Streptococcus gordonii

A disulfide bond is important for the correct folding of many proteins and is catalyzed by thiol-disulfide oxidoreductases (TDORs). These extracytoplasmic TDORs participate in diverse physiological processes. In Gram-positive bacteria, disulfide bond formation is poorly understood. Research in my laboratory has indicated that the oral bacterium Streptococcus gordonii is a model organism to study disulfide bond formation. Progress made include identification of a major TDOR (SdbA) that is needed for multiple phenotypes and identification of the redox partners (SdbB and CcdA2) of SdbA. The LONG-TERM GOAL of my research program is to elucidate the mechanisms of disulfide bond formation in Gram-positive bacteria. The SHORT-TERM GOALS will focus on the characterization of the disulfide bond formation and isomerization pathways in S. gordonii. The 3 objectives are: (1) Further investigations of the SdbA oxidative pathway; (2) Identification of additional natural substrates of SdbA; (3) Characterization of the disulfide isomerization pathway. For objective (1), the final electron acceptor(s) and other components of the SdbA-SdbB-CcdA2 pathway will be identified via transposon mutagenesis. Standard techniques will be used to study the identified gene(s) and its role in the pathway. For objective (2), the natural substrates of SdbA will be identified using a combination of in silico and proteomic approaches. Secreted and membrane-associated proteins with 2 or more cysteines will be identified from the S. gordonii proteome. In parallel, extracellular and cell surface proteins will be obtained via fractionation from the single cysteine SdbAC89A point mutant, which produced a number of mixed disulfide complexes. The complexes will be isolated using anti-SdbA antibody in affinity chromatography and identified by mass spectrometry. Proteins related to phenotypes mentioned above will be further analyzed to verify that they are substrates of SdbA. For objective (3), we showed that SdbB has disulfide isomerase activity. The work is to identify the partner and natural substrates of SdbB and the role of the isomerization pathway in cellular processes. Mutagenesis will be employed to investigate the functions of SdbB and its partner. SdbB substrates will be identified using immuno-affinity chromatography and proteomic approach similar to objective (2). SIGNIFICANCE of the work. The results will provide understanding to the mechanism of disulfide bond formation and cellular processes in the oral commensal S. gordonii. The knowledge gained is of fundamental importance to the understanding of adaptation and survival of oral bacteria. In the long-term, the knowledge will form the foundation of understanding how many of the disulfide bond-containing virulence factors (e.g. superantigens) are formed in Gram-positive pathogens.