Abstract:
The proteins AdeR (response regulator) and AdeS (histidine kinase sensor) play a crucial role in regulating the efflux pump, which is responsible for removing antibiotics from bacterial cells. This regulation occurs through a two-component regulatory system (TCS) involving autophosphorylation. Upon receiving external signals, AdeS undergoes autophosphorylation, leading to the activation of AdeR, which in turn initiates the expression of target genes related to the efflux pump. In this study, both enzymes from the reference strain of Acinetobacter baumannii (ATCC19606) and antibiotic resistant strains (H1074 and G560) were cloned and expressed in E. coli. The enzymes were purified using an immobilized metal ion affinity chromatography (IMAC) column, utilizing a C-terminal His-tag. To investigate the interaction characteristics between the antibiotic resistant strains and the reference strain, several procedures were employed, including electrophoretic mobility shift assay (EMSA), fluorescence resonance energy transfer (FRET), isothermal titration calorimetry (ITC), and enzymatic activity assay. The EMSA results demonstrated that both the reference and antibiotic resistant strains were capable of binding to the intercistronic DNA. The FRET analysis indicated that the mixing of AdeR labeled with Cy3 and AdeS labeled with Cy5 exhibited a higher intensity at 485 nm compared to the mixing of Cy3 and Cy5 alone. However, no FRET signal indicating binding between the donor and acceptor was detected. Moreover, the ITC results did not detect any binding between AdeR and DNA or between AdeR and AdeS. The enzymatic activity assay found that AdeS has kinase activity and ATP hydrolysis. In this study, demonstrate the ability of AdeR to interact with the intercistronic DNA. And suggest the occurrence of potential interactions between AdeR and AdeS. However, the exact binding affinity could not be determined through ITC. Additionally, the FRET experiments did not provide conclusive evidence of protein-protein interactions. Further investigations are warranted to understand the effects of variations in amino acids on the affinity of AdeR for DNA and the affinity of AdeR and AdeS in controlling efflux pump expression. These results have implications for manipulating protein interactions in this system to minimize drug resistance in A. baumannii.
