Characterization of the sensor domain of QseE histidine kinase from Escherichia coli

https://doi.org/10.1016/j.pep.2016.06.012Get rights and content

Highlights

  • The structural properties of the sensor domain of QseE histidine kinase.

  • CD and NMR data showed that the protein is a helical structure at pH 5.0 and 7.4.

  • MALS data confirmed that the protein is a monomer at pH 5.0 and 7.4.

Abstract

In enterohemorrhagic Escherichia coli (EHEC), the QseEF two-component system causes attaching and effacing (AE) lesion on epithelial cells. QseE histidine kinase senses the host hormone epinephrine, sulfate, and phosphate; it also regulates QseF response regulator, which activates LEE gene that encodes AE lesion. In order to understand the recognition of ligand molecules and signal transfer mechanism in pathogenic bacteria, structural studies of the sensor domain of QseE of Escherichia coli should be conducted. In this study, we describe the overexpression, purification, and structural and biophysical properties of the sensor domain of QseE. The fusion protein had a 6×His tag at its N-terminus; this protein was overexpressed as inclusion bodies in E. coli BL21 (DE3). The protein was denatured in 7M guanidine hydrochloride and refolded by dialysis. The purification of the refolded protein was carried out using Ni-NTA affinity column and size-exclusion chromatography. Thereafter, the characteristics of the refolded protein were determined from NMR, CD, and MALS spectroscopies. In a pH range of 7.4–5.0, the folded protein existed in a monomeric form with a predominantly helical structure. 1H-15N HSQC NMR spectra shows that approximately 93% backbone amide peaks are detected at pH 5.0, suggesting that the number of backbone signals is sufficient for NMR studies. These data might provide an opportunity for structural and functional studies of the sensor domain of QseE.

Introduction

Quorum sensing is also cell-to-cell communication in bacteria. It is a critical process for cell survival as it protects the cells from external environment through biofilm formation, virulence, and antibiotic resistance [1], [2], [3]. In this process, there is sensing of small molecules, such as oligopeptides in Gram-positive bacteria; AI-1 (autoinduer-1) and AI-2 (autoinducer-2), which are N-Acyl Homoserine Lactones (AHL) found in both Gram-negative and Gram-positive bacteria; and AI-3 (autoinducer-3) in Gram-negative bacteria [1]. AI-1 is synthesized by LuxI and released into the surrounding environment. When the population density of bacteria is high, the local concentration of AI-1 increases in the environment. Consequently, AI-1 diffuses into the cell. Then, LuxR easily senses AI-1 in the cell and activates the luciferase operon [4], [5], [6]. In Escherichia coli and Salmonella typhimurium, AI-2 is created from LuxS-derived 4,5-dihydroxy-2,3-pentanedione (DPD). Then, it is imported into the cell by LsrABC transporters. Finally, it is phosphorylated by LsrK. The phosphorylated AI-2 interacts with the transcriptional repressor, LsrR, which activates lsr operon [7]. In Vibrio spp, AI-2 (a furanosyl borate diester) binds with LuxP in the periplasm: the resultant complex interacts with the histidine kinase LuxQ. Consequently, there is phosphorylation of conserved histidine of LuxQ. The phosphate group is transferred to the response regulator LuxO via the LuxQ/LuxO phoshporyl cascade [1]. Recently, it has been proved that QseBC and QseFE are the two component systems that are involved in bacterial quorum sensing and regulation of virulence factors in pathogenic bacteria, such as enterohemorrhagic E. coli (EHEC) O157:H7 and salmonella enterica serovar Typhimurium [8], [9], [10], [11], [12]. QseC HK senses the auto inducer 3 (AI-3), epinephrine, and norepinephrine of host hormones. Its cognate response regulator QseB regulates virulence factors, including the locus of enterocyte effacement (LEE) genes; these genes cause intestinal colonization by stimulating the formation of ‘attaching and effacing’ (AE) lesions, flagellar, and motility genes [13]. The sensor domain of QseE not only senses epinephrine hormone but also phosphate and sulfate ions. Thereafter, it activates its response regulator QseF [9], [10], [12], which subsequently regulates the transcription of virulence factor genes, such as sipA and sopB genes; these genes are involved in the invasion of epithelial cells and espFu gene, driving the polymerization of actin during AE lesion formation [11], [12]. This indicates that the sensor domains of QseC and QseE may be antimicrobial drug targets that block bacterial quorum sensing and virulence factors [14]. Recently, we have successfully elucidated the structural characteristics of the sensor domain of QseC in solution state [15]. In this study, we produced and characterized the sensor domain of QseE of E. coli K12 strain, which has 99% sequence identity from that of EHEC O157:H7 strain. Thus, this study provides a foundation for structural and functional studies that focus on the sensor domains of QseE histidine kinases.

Section snippets

Cloning and expression of the sensor domain of QseE

The DNA sequence of the sensor domain of QseE (resiudes Q35-Q173) was amplified from genomic DNA of E. coli K12 using the polymerase chain reaction (PCR): 5′-CGCCATATGCAAAGCCTGAATGCGCTTAGCG-3′ was used as the forward primer, while 5′-CGCCTCGAGTCATTGCCCACGTTCGGCGATTTCA-3′ was used as the reverse primer. The PCR fragment was cloned into pET28a (+) vector using T4 ligase and the restriction enzymes: Nde I and Xho I. The plasmid encoded the QseE sensor domain, including 6×His-tag at its N-terminus;

Domain organization of QseE and secondary structure prediction of its sensor domain

Domain analysis was conducted using SMART (Simple Modular Architecture Research Tool) service. The results of domain analysis indicated that full length QseE consists of a periplasmic sensor domain (residues 35–174) and a kinase domain. The periplasmic sensor domain is located between two transmembrane helices (residues 15–34 and 175–197), while the kinase domain is composed of two subdomains (His Kinase A (phosphoacceptor) domain and ATP binding CA domain) (Fig. 1A). The secondary structure

Discussion

In pathogenic bacteria, cell-to-cell communication occurs through the recognition of signaling molecules in the host environment; this communication is essential for regulating the virulence factors of bacteria. In particular, QseBC and QseFE are the two component systems that sense host hormones: epinephrine and/or norepinephrine. Furthermore, QseBC and QseFE regulate the expression of virulence genes in pathogenic bacteria, such as EHEC and Salmonella enterica serovar Typhimurium [9], [10],

Acknowledgments

This research was supported by a Korea University Grant and the National Research Foundation of Korea (NRF) grant funded by the Korea government(Ministry of Science, ICT and Future Planning) (No. NRF-2014M3A9D9069710, NRF-2013M3A6A4045160, and NRF-2014R1A4A1007304). KYH was supported by KU grants.

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