Abstract

According to the sit-and-wait hypothesis, long-term environmental survival is positively correlated with increased bacterial pathogenicity because high durability reduces the dependence of transmission on host mobility. Many indirectly transmitted bacterial pathogens, such as Mycobacterium tuberculosis and Burkhoderia pseudomallei, have high durability in the external environment and are highly virulent. It is possible that abiotic stresses may activate certain pathways or the expressions of certain genes, which might contribute to bacterial durability and virulence, synergistically. Therefore, exploring how bacterial phenotypes change in response to environmental stresses is important for understanding their potentials in host infections. In this study, we investigated the effects of different concentrations of salt (sodium chloride, NaCl), on survival ability, phenotypes associated with virulence, and energy metabolism of the lab strain Escherichia coli BW25113. In particular, we investigated how NaCl concentrations influenced growth patterns, biofilm formation, oxidative stress resistance, and motile ability. In terms of energy metabolism that is central to bacterial survival, glucose consumption, glycogen accumulation, and trehalose content were measured in order to understand their roles in dealing with the fluctuation of osmolarity. According to the results, trehalose is preferred than glycogen at high NaCl concentration. In order to dissect the molecular mechanisms of NaCl effects on trehalose metabolism, we further checked how the impairment of trehalose synthesis pathway (otsBA operon) via single-gene mutants influenced E. coli durability and virulence under salt stress. After that, we compared the transcriptomes of E. coli cultured at different NaCl concentrations, through which differentially expressed genes (DEGs) and differential pathways with statistical significance were identified, which provided molecular insights into E. coli responses to NaCl concentrations. In sum, this study explored the in vitro effects of NaCl concentrations on E. coli from a variety of aspects and aimed to facilitate our understanding of bacterial physiological changes under salt stress, which might help clarify the linkages between bacterial durability and virulence outside hosts under environmental stresses.

Conclusion

In this study, we investigated the influences of NaCl concentrations on growth patterns, phenotypes associated with virulence, and energy metabolism in wild-type Escherichia coli BW25113 and its two single-gene mutants ΔotsA and ΔotsB. The results indicated that elevated NaCl concentrations in the culture medium generally inhibited bacterial growth, biofilm formation, oxidative resistance, and motile ability in both wild-type strain and trehalose-deficient mutants. As for energy metabolism, it was confirmed that trehalose was preferred under hyper-saline conditions than glycogen in E. coli BW25113, while glucose uptake was significantly inhibited at higher NaCl levels. As for the E. coli ΔotsA and ΔotsB, trehalose was completely abolished in the two mutated strains. However, when compared E. coli wild-type strain with its two mutants, it was shown that inactivation of trehalose synthesis pathway facilitated biofilm formation at low salinity level. Further transcriptomic analysis revealed the significantly up- and down-regulated genes that were responsible for E. coli responses in high salinity condition. Hub genes and enriched pathways were also identified for general understanding of E. coli survival strategy under osmotic pressure. Although representative phenotypes associated with pathogenic characteristics in E. coli were negatively impacted by hyperosmotic pressure in this study, transcriptomic analysis revealed that gene expressions related with salt response and virulence were actually inter-connected. Thus, this study does not necessarily disapprove the sit-and-wait hypothesis since evolution involves long-term interactions with and adaptations to environment. Further studies may need to examine the phenotype changes of E. coli after long-term adaptation to hyperosmotic conditions, investigating specific pathways and gene clusters by experimental methodologies at molecular level for a better understanding of E. coli adaptations to abiotic stresses.