Molecular regulatory mechanisms of Escherichia coli O157:H7 in response to ultrasonic stress revealed by proteomic analysis

https://doi.org/10.1016/j.ultsonch.2019.104835Get rights and content

Highlights

  • Proteome revealed molecular regulatory mechanisms under ultrasonic stress.

  • Ultrasonic filed influenced various metabolic pathways in E. coli O157:H7 cells.

  • RpoS protein, MS channels, SOS response protein RecA were up-regulated.

  • Free radicals might enter into cells via activated mechanosensitive channels.

  • All-or-nothing effect might due to destruction of crucial cell defensive systems.

Abstract

The antimicrobial effects of ultrasonic filed have been studied for years at the phenotypic level, but there is little research to reveal the molecular regulatory mechanisms underlying the phenotypes. In this study, isobaric tag for relative and absolute quantification (iTRAQ) proteome was applied to analyze the regulatory networks of Escherichia coli O157:H7 in response to ultrasonic stress in whole-genome scale. A total of 1856 differentially expressed proteins were identified, of which 1141 were significant up-regulated and 715 down-regulated compared with live control cells. The comprehensive proteome coverage analysis showed that ultrasonic filed influenced various metabolic pathways in Escherichia coli O157:H7 cells. The ultrasound-induced up-regulation of global stress response regulator RpoS, bacterial mechanosensitive channels and SOS response protein RecA were described from the molecular level for the first time. In addition, we proposed a possible action mechanism that the free radicals produced by acoustic cavitation might enter into cells via the activated mechanosensitive channels, leading to the elevated intracellular ROS level and subsequent cell death. Last but not the least, we illustrated the all-or-nothing phenomenon of power ultrasound might due to the destruction of crucial cell defensive systems, including heat shock proteins and oxidative response regulators. These new findings can let us understand the ultrasonic effects more deeply and will contribute to this area.

Introduction

The study of ultrasound and its antimicrobial mechanisms on microorganisms have been the focus of active research for many years. Recent studies showed that acoustic cavitation plays the key role in microbial inactivation [1], [2], [3]. The collapse of bubbles in transient cavitation creates localized hot spots, shock waves and liquid jets, accompanied with generation of hydrogen peroxide and free radicals [4], [5]. The stable oscillation of bubbles below cavitation threshold does not cause bubble collapse but still produces micro-streaming along with extensive shear forces. As for cells situated close to the cavitation field, the mechanical stresses can distort cells and lead to cell destruction completely, involving pore formation, cell membrane disruption and the release of cytoplasm contents [6], [7]. While for cells located outside the area of cavitation, the hydrogen peroxide and free radicals were able to lead to cell oxidative damage. These chemical effects were considered to be the reason for the DNA damage in vitro. Although free radical production is hard to be detected in vivo due to the extremely low range, we have observed elevated levels of ROS in cells in earlier study, which might trigger programmed cell death [6]. As we can see, most efforts were put into the researches on figuring out how acoustic energy act on microbes, but the microbial responses to cavitation have been overlooked.

In order to survive, bacteria have evolved many complicated regulatory networks to counteract the stressors and repair the damages. Besides the natural stress conditions like cold, heat, oxidation, limited nutrition, adverse osmotic pressure and pH [8], [9], all sorts of preservation techniques, including the physical processing such as plasma, UV radiation, pulsed electric fields and high hydrostatic pressure can also put pressure on bacterial cells and trigger diverse stress responses [10], [11]. It was suggested that the global stress response regulator RpoS is significant for the cellular defensive response to the cold atmospheric plasma [12]. Vanlint et al. reported that cAMP/CRP homeostasis also affected the resistance of extreme high hydrostatic pressure in Escherichia coli O157:H7 independently of increased RpoS activity [13]. UV-specific response in Sulfolobus solfataricus showed that the well-known SOS response in bacteria was not involved, but shed light on novel strategies for DNA repair [14]. Alvarez-Ordóñez summarized that main aspects regarding the adaptive response involved cell defensive systems, cell membranes modification, shock response proteins synthesis, sensing mechanisms and homeostatic and repair systems [15].

To our knowledge, the microbial response to ultrasound has not been investigated in whole-genome scale yet. Therefore, this research was aimed at comprehensively assessing the ultrasonic-induced microbial response of Escherichia coli O157:H7 using iTRAQ-based proteomic analysis, and determining the molecular regulatory mechanisms that underlie the phenotypes that have been observed in our previous studies.

Section snippets

Bacteria preparation

Escherichia coli O157:H7 used in this study was obtained from China Center of Industrial Culture Collection. The cultures were stored at −80 °C refrigerator in nutrient broth with 50% glycerol. The strain was incubated in nutrient broth at 37 °C with shaking at 150 rpm to achieve the concentration of 9 log CFU/mL. Bacteria were centrifuged at 2500g for 10 min and then collected after washing by 0.85% (w/v) sterile saline solution.

Ultrasonic exposure

The experiments were performed in triplicate using an ultrasonic

Protein identification

Control and ultrasound-treated samples were prepared for iTRAQ proteomics. One was treated by ultrasound and the other was untreated as a control group. It showed that 326,938 spectra were produced, 30,807 peptides and 3015 proteins were confirmed with 1% FDR. The molecular masses of the identified proteins mainly ranged between 10 and 60 kDa. CV was used to evaluate the reproducibility, which was defined as the ratio of the standard deviation to the mean. The mean CV in this experiment was

Discussion

Based on the proteomic analysis, we further discussed the DEPs of E. coli O157:H7 involved in stress responses under ultrasonic field. Table 1 shows the statistics and classification of microbial response related DEPs, mainly including general stress response, mechanical stress response, DNA damage response, heat shock response and oxidative stress response. What’s more, Fig. 7 illustrates those regulatory pathways of E. coli O157:H7 in response to ultrasonic stress.

Conclusion

In this research, we investigated the molecular regulatory networks of E. coli O157:H7 in response to ultrasonic filed using the iTRAQ proteomic methods. Different stress response systems were found differentially expressed. The general stress response protein, RpoS, was accumulated. Bacterial mechanosensitive channels (MscL, MscS, MscM, MscK) were activated presumably by the mechanical tension on the membrane. In addition, the SOS response was also activated accompanied by the up-regulated

Acknowledgement

This study is supported by the National Key Research and Development Program of China (2016YFD0400301).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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