Quantitative proteomic analysis of Edwardsiella tarda in response to oxytetracycline stress in biofilm

Abstract

Edwardsiella tarda is a virulent fish pathogen that causes extensive economic losses in the aquaculture industry worldwide. The antibiotic resistance status of E. tarda is high, especially in the biofilm status; however, the mechanisms underlying its resistance remain largely unknown. In this study, isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to compare the differential expression of E. tarda in response to oxytetracycline (OXY) stress in biofilm. Additional bioinformatics analysis demonstrated an increasing abundance of translation-related proteins, especially ribosomal subunits, and a decreasing abundance of key metabolic pathways underlying the adaptation of E. tarda to OXY in biofilm. We performed Western blotting and quantitative PCR (qPCR) analyses to validate selected proteomics results, and measured enzyme activity to verify the antibiotic resistance functions of central metabolic pathways. In addition, we examined the antibiotic susceptibility of a mutant of an NADP-dependent malic enzyme (MaeB), which is involved in the bacterial tricarboxylic acid cycle, and found significantly increased resistance to OXY in biofilm. Our findings demonstrate the importance of central metabolic pathways in the antibiotic resistance of E. tarda to bacterial biofilms and provide insight into the prevention of this resistance, which would aid in disease control.

Biological significance: The antibiotics resistance mechanisms in E. tarda have been well documented recently; however, its response to antibiotics in biofilms remains elusive. Our current study is the first exploratory report investigating this aspect via an iTARQ-based quantitative proteomics method. Several important proteins, related processes, and metabolic pathways were found to be involved in OXY fitness in biofilm status. Most importantly, the depletion of the maeB gene decreased the susceptibility of E. tarda to OXY indicating the important role of central metabolic pathways in antibiotics resistance in biofilm.

Keywords: Antibiotic resistance mechanism; Biofilm; Edwardsiella tarda; Quantitative proteomics.