The role of PhoP/PhoQ two component system in regulating stress adaptation in Cronobacter sakazakii

doi:10.1016/j.fm.2021.103851

Food Microbiology

Volume 100, December 2021, 103851

https://doi.org/10.1016/j.fm.2021.103851 Get rights and content

Highlights

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PhoP/PhoQ enhanced C. sakazakii growth under various conditions.
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PhoPQ mutant exhibited reduced tolerance to different environmental stress.
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RNA-seq analysis revealed potential regulons of PhoP/PhoQ in C. sakazakii.

Abstract

Cronobacter sakazakii is an opportunistic foodborne bacterial pathogen that shows resistance to multiple stress conditions. The PhoP/PhoQ two component system is a key regulatory mechanism of stress response and virulence in various bacteria, but its role in C. sakazakii has not been thoroughly studied. In this study, we found the PhoP/PhoQ system in C. sakazakii ATCC BAA-894 enhanced bacterial growth in conditions with low Mg²⁺, acid pH, and the presence of polymyxin B. Moreover, the ΔphoPQ strain significantly reduced survival following exposure to heat, high osmotic pressure, oxidative or bile salts compared with WT strain. Furthermore, the RNA-seq analysis indicated that 1029 genes were upregulated and 979 genes were downregulated in ΔphoPQ strain. The bacterial secretion system, flagella assembly, beta-Lactam resistance and two-component system pathways were significantly downregulated, while the ABC transporters and microbial metabolism in diverse environments pathways were upregulated. qRT-PCR analysis further confirmed that twelve genes associated with stress tolerance were positively regulated by the PhoP/PhoQ system. Therefore, these findings suggest that the PhoP/PhoQ system is an important regulatory mechanism for C. sakazakii to resist various environmental stress.

Introduction

Cronobacter sakazakii (C. sakazakii) is an opportunistic Gram-negative foodborne pathogen that cause severe infections in neonates and infants, such as meningitis, bacteremia, and necrotizing enterocolitis (Du et al., 2018). The organisms have been isolated from various foods and environments, with powdered infant formula (PIF) being considered one of the most common vehicles of transmission (Lou et al., 2019; Vojkovska et al., 2016). The ability of foodborne pathogens to resist many environmental stresses including heat, acid, osmotic press, dehydration, bile salts and other hostile conditions is responsible for their survival and spread (Caubilla-Barron et al., 2007; Lang et al., 2018; Osaili and Forsythe, 2009).

C. sakazakii shows distinctive resistance to adverse environmental conditions compared with other Enterobacteriaceae (Jaradat et al., 2014). Drying is an extremely arduous condition of osmotic pressure, which makes cells difficult to survive, and polyhydroxy compounds such as trehalose can replace the shell of the water around the polymer, thereby protecting the cells from damage (Leslie et al., 1995).Breeuwer et al. (2003) also found that the cumulation of trehalose in cells confers C. sakazakii with strong resistance to desiccation. During the production of PIF, high temperature treatment is generally adopted for controlling microbial contamination. Since C. sakazakii has strong ability to withstand high temperature and drying, there is still a high residual risk after thermal dehydration and pasteurization (Chen et al., 2019).

Two-component systems (TCSs), a primary form of bacterial signal transduction, can regulate the ability of bacteria to sense and respond to the external environment. TCSs usually consists of two parts: histidine kinase (HK) and response regulator (RR). HK can autophosphorylate according to the presence of the signal, and then the phosphate group can be transferred to response regulator protein, which then binds to the gene promoter region to regulate downstream gene expression (Jacob-Dubuisson et al., 2018). Many TCSs have been reported in bacteria, such as PhoP/PhoQ, PmrA/PmrB, CpxA/CpxR. TCSs can play a role alone to regulate the survival of bacteria in adverse environments, or interact with other TCSs to act synergistically (Carabajal et al., 2019; Huang et al., 2016; Kox et al., 2000).

The PhoP/PhoQ system is a regulatory mechanism of stress response and virulence control in many Gram-negative bacteria. When the PhoQ is activated by signals, it autophosphorylate and transfer the phosphate group to PhoP, thereby regulating the expression of a series of downstream genes, such as Mg²⁺ homeostasis, stress tolerance, and toxicity (Yoshitani et al., 2019). In Salmonella, the PhoP/PhoQ controls the expression of key phenotypes that determine infection process (Carabajal et al., 2019). In Shigella flexneri, the PhoP/PhoQ system regulates the expression of its virulence genes and its tolerance to low Mg²⁺, acid pH, and polymyxin B (Lin et al., 2017). Although PhoP/PhoQ has been proved to have extensive regulatory effects in Enterobacteriaceae such as Salmonella, E. coli, the regulatory role of PhoP/PhoQ in C. sakazakii has been rarely studied.

The aim of this study was to evaluate the key role of the PhoP/PhoQ system in regulating stress tolerance of C. sakazakii. The phoPQ gene deletion mutant and complemented strains were constructed. The effects of phoPQ gene deletion on the resistance of C. sakazakii to multiple environmental stresses were evaluated, including low Mg²⁺, acidic pH, polymyxin B, acid, heat, bile salts, and osmotic conditions. Additionally, RNA-sequencing and qRT-PCR were performed to decipher the regulons of phoPQ in C. sakazakii and to explore possible mechanisms by which this TCS play its roles in stress tolerance.

Section snippets

Bacterial strains and plasmids

The bacterial strains and plasmids used in this study are listed in Table 1. C. sakazakii strains and E. coli strains were grown in Luria-Bertani (LB) medium at 37 °C. Antibiotics were used at the following concentrations where necessary: chloramphenicol (Cm) at 20 μg/mL; nalidixic acid (NA) at 32 μg/mL.

Construction of the phoPQ mutant and complemented strain

The phoPQ deletion mutant of C. sakazakii BAA-894/NA was generated by sacB-based two-step allelic exchange as previously described (Hmelo et al., 2015). Briefly, the upstream and downstream

Construction of the phoPQ gene mutant and complemented strain

The mutant strain ΔphoPQ was confirmed by PCR using the primers phoPQ-UP-F/phoPQ-DOWN-R (Fig. 1A) and phoPQ-IN-F/R (Fig. 1B). And the complemented strain ΔphoPQ^C was verified by PCR using the primers phoPQ-IN-F/R (Fig. 1C).

The PhoP/PhoQ system regulates the resistance of C. sakazakii to low Mg²⁺, acid pH and polymyxin B

To analyze the role of PhoP/PhoQ system in C. sakazakii ability to adapt to low Mg²⁺ and acidic pH, the growth curves of WT, ΔphoPQ and ΔphoPQ^C strains under different Mg²⁺ concentrations and pH were measured (Fig. 2A–G), and the growth kinetic parameters including maximum

Discussion

The PhoP/PhoQ system is the first identified regulatory system that uses extracellular Mg²⁺ as the major signal (Groisman, 2001). The system exists in numerous pathogenic bacteria, such as Salmonella, E. coli, Shigella, Serratia, the sensor PhoQ can response to divalent cations, acidic pH and cationic antimicrobial peptides (Barchiesi et al., 2012; Carabajal et al., 2020; Lin et al., 2017; Zhuge et al., 2018). Fowler et al. reported that the mechanisms involving the PhoP/PhoQ system were used

Conclusion

In conclusion, the PhoP/PhoQ system is involved in the regulating the tolerance of C. sakazakii to various environment stress including low Mg²⁺, acidic pH, polymyxin B, heat, hyperosmotic, oxidative and bile salts stress. Deletion of the phoPQ gene in C. sakazakii BAA-894 led to a significant reduction in its tolerance to different environmental stress. RNA-seq analysis provided a general picture for the regulons of PhoP/PhoQ system in C. sakazakii. Both RNA-seq and qRT-PCR analysis showed

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (31772084), and LiaoNing Revitalization Talents Program (XLYC1807220). Shaanxi Key Research and Development project 2019SF-259, the Fundamental Research Funds for the Central Universities (2452017146 and 2452017228).

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The role of PhoP/PhoQ two component system in regulating stress adaptation in Cronobacter sakazakii

Highlights

Abstract

Introduction

Section snippets

Bacterial strains and plasmids

Construction of the phoPQ mutant and complemented strain

Construction of the phoPQ gene mutant and complemented strain

The PhoP/PhoQ system regulates the resistance of C. sakazakii to low Mg2+, acid pH and polymyxin B

Discussion

Conclusion

Declaration of competing interest

Acknowledgements

Food Res. Int.

Food Chem.

Food Contr.

Food Microbiol.

Int. J. Food Microbiol.

Int. J. Food Microbiol.

Food Microbiol.

Int. J. Food Microbiol.

Int. J. Food Microbiol.

Food Contr.

Int. J. Food Microbiol.

Polymorphisms in rpoS and stress tolerance heterogeneity in natural isolates of Cronobacter sakazakii

Appl. Environ. Microbiol.

Differential expression analysis for sequence count data

Genome Biol.

Survival and environmental stress resistance of Cronobacter sakazakii exposed to vacuum or air packaging and stored at different temperatures

Front. Microbiol.

The PhoP/PhoQ system and its role in Serratia marcescens pathogenesis

J. Bacteriol.

A high-throughput system to identify inhibitors of Candidatus Liberibacter asiaticus transcription regulators

Proc. Natl. Acad. Sci. U. S. A.

Desiccation and heat tolerance of Enterobacter sakazakii

J. Appl. Microbiol.

Quinazoline-based anti-virulence compounds selectively target Salmonella PhoP/PhoQ signal transduction system

Antimicrob agents ch 64

PhoQ is an unsaturated fatty acid receptor that fine-tunes Salmonella pathogenic traits

Sci. Signal.

Genotypic and phenotypic analysis of Enterobacter sakazakii strains from an outbreak resulting in fatalities in a neonatal intensive care unit in France

J. Clin. Microbiol.

Structural basis for inhibition of a response regulator of σS stability by a ClpXP antiadaptor

Genes Dev.

Characterization of the desiccation tolerance of Cronobacter sakazakii strains

Front. Microbiol.

Increased mistranslation protects E. coli from protein misfolding stress due to activation of a RpoS-dependent heat shock response

FEBS Lett.

Stress tolerant virulent strains of Cronobacter sakazakii from food

Biol. Res.

Safe Preparation, Storage and Handling of Powdered Infant Formula-Guidelines

Analysis of the role of the Cronobacter sakazakii ProP homologues in osmotolerance

Gut Pathog.

Antibiotic and desiccation resistance of Cronobacter sakazakii and C. malonaticus isolates from powdered infant formula and processing environments

Front. Microbiol.

Decoding a Salmonella typhi regulatory network that controls typhoid toxin expression within human cells

Cell Host Microbe

The PhoP/PhoQ system regulates the resistance of C. sakazakii to low Mg²⁺, acid pH and polymyxin B

Structural basis for inhibition of a response regulator of σ^S stability by a ClpXP antiadaptor