ABSTRACT: Salmonella enterica subsp. enterica serovar Enteritidis is able to adapt to sublethal concentrations of ethanol, which subsequently induce tolerance of this pathogen to normally lethal ethanol challenges. This work aims to elucidate the underlying ethanol adaptation mechanisms of S Enteritidis by proteomic and mutagenic analyses. The global proteomic response of S Enteritidis to ethanol adaptation (5% ethanol for 1 h) was determined by isobaric tags for relative and absolute quantification (iTRAQ), and it was found that a total of 138 proteins were differentially expressed in ethanol-adapted cells compared to nonadapted cells. A total of 56 upregulated proteins were principally associated with purine metabolism and as transporters for glycine betaine, phosphate, d-alanine, thiamine, and heme, whereas 82 downregulated proteins were mainly involved in enterobactin biosynthesis and uptake, the ribosome, flagellar assembly, and virulence. Moreover, mutagenic analysis further revealed the functions of two highly upregulated proteins belonging to purine metabolism (HiuH, 5-hydroxyisourate hydrolase) and glycine betaine transport (ProX, glycine betaine-binding periplasmic protein) pathways. Deletion of either hiuH or proX resulted in the development of a stronger ethanol tolerance response, suggesting negative regulatory roles in ethanol adaptation. Collectively, this work suggests that S Enteritidis employs multiple strategies to coordinate ethanol adaptation.IMPORTANCE Stress adaptation in foodborne pathogens has been recognized as a food safety concern since it may compromise currently employed microbial intervention strategies. While adaptation to sublethal levels of ethanol is able to induce ethanol tolerance in foodborne pathogens, the molecular mechanism underlying this phenomenon is poorly characterized. Hence, global proteomic analysis and mutagenic analysis were conducted in the current work to understand the strategies employed by Salmonella enterica subsp. enterica serovar Enteritidis to respond to ethanol adaptation. It was revealed that coordinated regulation of multiple pathways involving metabolism, ABC transporters, regulators, enterobactin biosynthesis and uptake, the ribosome, flagellar assembly, and virulence was responsible for the development of ethanol adaptation response in this pathogen. Such knowledge will undoubtedly contribute to the development and implementation of more-effective food safety interventions.
