Determining the Interactions of QseC and YgiW With CatFe

Objectives: During a periodontitis infection, neutrophils release catecholamines, thus A. actinomycetemcomitans may take advantage of this CAT rich environment to acquire an essential nutrient, iron (Fe). The QseBC two-component system (TCS), is essential for biofilm growth and virulence of Aa, and facilitates the transport of iron Aa. Further, QseBC is expressed along with a second protein, YgiW. However, there is no known function for this protein. Thus, our objective is to examine the interaction of QseC and YgiW with catecholamines and iron.
Methods: Proteins were expressed using previously constructed plasmids. Following expression, proteins were identified by SDS-PAGE and Western Blot using specific antibodies. Subsequently, protein purification was accomplished by affinity column chromatography and a two-chamber dialysis system. Proteins were quantified by NanoDrop. The interaction of each protein with catecholamines and/or iron was evaluated using equilibrium dialysis and measuring CAT and Fe levels using ELISA and a colorimetric assay, respectively.
Results:
YgiW was present in the soluble fraction after cell lysis, while a significant portion of expressed QseC remained associated with the cellular debris after lysis. After an additional round of suspension and lysis, the yield of QseC in the soluble fraction increased substantially. Both proteins were readily purified by affinity chromatography. The conditions for equilibrium dialysis were optimized to determine binding of CAT and Fe to QseC and/or YgiW. If YgiW interacts with CAT, Fe or CAT-Fe, it is likely that these ligands first activate YgiW, which in turn could activate QseC by interacting with its periplasmic domain. Conversely, the ligands could only interact with the periplasmic domain of QseC, requiring further study of the role that YgiW.
Conclusions:
The QseC and YgiW proteins have been purified and conditions for equilibrium dialysis have been optimized to begin determining the interaction kinetics of catecholamines and iron.