Characterization of ampicillin-stressed proteomics and development of a direct method for detecting drug-binding proteins in Edwardsiella tarda.

ABSTRACT

Antibiotic-resistant Edwardsiella tarda poses a severe challenge to aquaculture. An understanding for antibiotic-resistant mechanisms is crucial to control the disease. The present study characterizes E. tarda ampicillin-stressed proteome and shows the importance of energy metabolism including the TCA cycle and glycolysis/gluconeogenesis in the antibiotic resistance. Further combination with antibiotic measurement develops a new method for identification of antibiotic-binding proteins out of differential abundances of proteins and results in determination of ETAE_0175 and ETAE_3367 as ampicillin-binding proteins in E. tarda. Genes of the two proteins are cloned and recombinant proteins are purified for validation of antibiotic-binding capability. Results show that higher binding capability is detected in ETAE_3367 than ETAE_0175. Out of the two proteins, ETAE_3367 is first reported here to be an antibiotic-binding protein, while ETAE_0175 homology in other bacteria has been shown to bind with other antibiotics. Bioinformatics analysis shows that ETAE_3367 may closely interact with aceF and sucA belonging to the TCA cycle and glycolysis/gluconeogenesis, respectively. These results indicate that energy metabolism contributes to ampicillin resistance in E. tarda and a new method to identify antibiotic-binding proteins is developed. These findings highlight the way to an understanding of antibiotic-resistant mechanisms in content of antibiotic-binding proteins. BIOLOGICAL SIGNIFICANCE: Our data characterizes Edwardsiella tarda ampicillin-stressed proteome and shows the importance of energy metabolism including the TCA cycle and glycolysis/gluconeogenesis in the antibiotic resistance. Furthermore, a new method based 2-DE proteomics is developed for identification of antibiotic-binding proteins, which results in determination of ETAE_0175 and ETAE_3367 as ampicillin-binding proteins in E. tarda. ETAE_3367 is closely interacted with proteins of the TCA cycle and glycolysis/gluconeogenesis, suggesting the drug-resistant mechanism.