Proteomics study unveils ROS balance in acid-adapted Salmonella Enteritidis.

Salmonella Enteritidis is a major cause of foodborne gastroenteritis and is thus a persistent threat to global public health. The acid adaptation response helps Salmonella survive exposure to gastric environment during ingestion. In a previous study we highlighted the damage caused to cell membrane and the regulation of intracellular reactive oxygen species (ROS) in S. Enteritidis. In this study, we applied both physiologic and iTRAQ analyses to explore the regulatory mechanism of acid resistance in Salmonella. It was found that after S. Enteritidis was subject to a 1 h period of acid adaptation at pH 5.5, an additional 1 h period of acid shock stress at pH 3.0 caused less Salmonella cell death than in non-acid adapted Salmonella cells. Although there were no significant differences between adapted and non-adapted cells in terms of cell membrane damage (e.g., membrane permeability or lipid peroxidation) after 30 min, intracellular ROS level in acid adapted cells was dramatically reduced compared to that in non-acid adapted cells, indicating that acid adaption promoted less ROS generation or increased the ability of ROS scavenging with little reduction in the integrity of the cell membrane. These findings were confirmed via an iTRAQ analysis. The adapted cells were shown to trigger incorporation of exogenous long-chain fatty acids into the cellular membrane, resulting in a different membrane lipid profile and promoting survival rate under acid stress. S. Enteritidis experiences oxidative damage and iron deficiency under acid stress, but after acid adaption S. Enteritidis cells were able to balance their concentrations of intracellular ROS. Specifically, SodAB consumed the free protons responsible for forming reactive oxygen intermediates (ROIs) and KatE protected cells from the toxic effects of ROIs. Additionally, acid-labile proteins released free unbound iron promoting ferroptotic metabolism, and NADH reduced GSSH to G-SH, protecting cells from acid/oxidative stress.

Hu S ，Yu Y ，Lv Z ，Shen J ，Ke Y ，Xiao X ... -  《-》

Membrane lipid composition and stress/virulence related gene expression of Salmonella Enteritidis cells adapted to lactic acid and trisodium phosphate and their resistance to lethal heat and acid stress.

This study evaluated the acid and heat resistance of Salmonella Enteritidis in simulated gastric fluid (pH 2.0) and during thermal treatment (54-60 °C), respectively, after adaptation to lactic acid (LA) or trisodium phosphate (TSP) at various pHs (pH 5.3-9.0). The changes in membrane lipid composition and expression levels of RpoS and RpoH were examined to elucidate their roles in bacterial stress resistance. Transcriptional profile of several virulence-related genes was also analyzed. Results showed that LA-adapted cells at pH 5.3 and 6.3 had higher acid and heat resistance than control cells and cells adapted to TSP at pH 8.3 and 9.0. LA-adapted cells had the lowest ratio of unsaturated to saturated fatty acids, indicating that they might possess a less fluid membrane. It was observed that the expression levels of RpoH and RpoS were upregulated in TSP-adapted cells but not in LA-adapted cells. Thus, these results indicate that the increased acid and heat resistance of LA-adapted S. Enteritidis was possibly due to the decreased membrane fluidity instead of the upregulation of RpoS and RpoH. About 6.0, 2.1, and 2.46-fold upregulation of spvR, avrA, and hilA were observed in cells adapted to TSP at pH 9.0, except sefA that had its highest expression level in the control cells, indicating that the expression of these virulence genes highly depends on environmental conditions. This is the first study to show that the alteration in the cytoplasmic membrane rather than RpoS and RpoH plays a more crucial role in conferring greater acid and heat resistance on LA-adapted S. Enteritidis, thus providing a better understanding on the bacterial stress response to acidic conditions.

Yang Y ，Kadim MI ，Khoo WJ ，Zheng Q ，Setyawati MI ，Shin YJ ，Lee SC ，Yuk HG ... -  《-》

Influence of ethanol adaptation on Salmonella enterica serovar Enteritidis survival in acidic environments and expression of acid tolerance-related genes.

Cross-protection to environmental stresses by ethanol adaptation in Salmonella poses a great threat to food safety because it can undermine food processing interventions. The ability of Salmonella enterica serovar Enteritidis (S. Enteritidis) to develop acid resistance following ethanol adaptation (5% ethanol for 1 h) was evaluated in this study. Ethanol-adapted S. Enteritidis mounted cross-tolerance to malic acid (a two-fold increase in minimum bactericidal concentration), but not to acetic, ascorbic, lactic, citric and hydrochloric acids. The population of S. Enteritidis in orange juice (pH 3.77) over a 48-h period was not significantly (p > 0.05) influenced by ethanol adaptation. However, an increased survival by 0.09-1.02 log CFU/ml was noted with ethanol-adapted cells of S. Enteritidis compared to non-adapted cells in apple juice (pH 3.57) stored at 25 °C (p < 0.05), but not at 4 °C. RT-qPCR revealed upregulation of two acid tolerance-related genes, rpoS (encoding σS) and SEN1564A (encoding an acid shock protein), following ethanol adaptation. The relative expression level of the acid resistance gene hdeB did not change. The resistance phenotypes and transcriptional profiles of S. Enteritidis suggest some involvement of rpoS and SEN1564A in the ethanol-induced acid tolerance mechanism.

He S ，Cui Y ，Qin X ，Zhang F ，Shi C ，Paoli GC ，Shi X ... -  《-》

Ethanol Adaptation Strategies in Salmonella enterica Serovar Enteritidis Revealed by Global Proteomic and Mutagenic Analyses.

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.

He S ，Qin X ，Wong CWY ，Shi C ，Wang S ，Shi X ... -  《-》

Heat resistance of Salmonella Enteritidis under prolonged exposure to acid-salt combined stress and subsequent refrigeration.

Salmonella Enteritidis is a major foodborne pathogen exposed to various environmental and preservation stresses in the food chain. Because adaptive responses of viable bacterial cells in the presence of sublethal stress can induce cross-protection against different stresses, we investigated the heat resistance of Salmonella Enteritidis at 60 °C under prolonged exposure to acid-salt combined stress and subsequent refrigeration. Salmonella Enteritidis was grown in tryptic soy broth at four pH values (4.5, 5.4, 6.4, and 7.3) and four NaCl concentrations (0%, 1%, 2%, and 3%) at 37 °C for 24 h and then incubated at 4 °C for 0, 1, 4, or 7 days. For 0 and 1 day-refrigerated cultures, previous adaptation to single stresses (acid or salt stress) increased the heat resistance of Salmonella Enteritidis, resulting in increased D-values, whereas the combination of acid and salt stress reduced heat tolerance; acid stress played a more critical role in mediating this effect than salt concentration. To elucidate the related mechanisms, the expression levels of heat shock sigma factors (rpoH) and heat shock genes (dnaK and groEL) were analyzed and found to be associated with the heat resistance of Salmonella Enteritidis. The refrigeration period was negatively correlated (P < 0.01) with the D-value (r = -0.505) and with the transcript levels of rpoH (r = -0.654), dnaK (r = -0.652), and groEL (r = -0.645). Our findings demonstrated that acid-salt combined preservation techniques and subsequent refrigeration may prevent S. Enteritidis survival in heat-pasteurized food products caused by cross-protection of acid or salt adapted cells.

Kang IB ，Kim DH ，Jeong D ，Park JH ，Seo KH ... -  《-》